All the experiments were carried out according to the Norma Oficial Mexicana (NOM-062-ZOO-1999, 9.4.2.1.3.) for the care and handling of laboratory animals. The protocols were reviewed and approved by the Animal Care and Use Committee of the Benemerita Universidad Autonoma de Puebla, identification code: BERRSAL71, 18-05-2017. Experiments were carried out in male ZDF rats (3 months old) from Charles River Laboratories, California, U.S.A. Throughout the text, diabetic-obese ZDF rats (ZDF-Lepr^fa/fa) will be designated as OZDF rats, and lean controls, non-obese non-diabetic ZDF (ZDF-Lepr^+/+) as LZDF. The rats were kept at the University Animal Core Facilities under controlled environmental temperature and, exposed to light-dark cycles of 12 h, with ad libitum consumption of water and high energy diet, Purina 5,008 chow.

To determine if OZDF rats did indeed develop T2DM, 5 days prior to the experiment, a glucose tolerance test was performed. The animals were fasted for 6 h in metabolic cages with free access to water. Immediately after, a glucose dose (2 g/kg of weight) was administrated intraperitoneally. Two hours later, a blood sample was obtained by puncture of the caudal vein to measure glucose levels using the Accucheck^® system (Roche, Mannheim, Germany). The day of the experiment, we proceeded to measure the body mass (weight), length (distance from the tip of the nose to the base of the tail) and the abdominal circumference using a measuring tape.

The body mass index (BMI) was calculated using the following equation: B M I = B o d y   m a s s   g L e n g t h 2   c m 2

Finally, all the epididymal fat surrounding both testes was accurately removed and weighed.

Isolation and culture of rat VSMCs were carried out according to the protocol previously described by Berra-Romani et al. to isolate proliferating, non-contracting VSMCs (Berra-Romani et al., 2008). ZDF rats were anesthetized with intraperitoneal ketamine-xylazine solution, 0.2 mL per 100 g of weight. The thoracic aorta was dissected out and perfused with low-Ca²⁺ physiological salt solution (PSS1, see composition in Section 2.5). Under sterile conditions the artery was cleaned of fat and connective tissue and then incubated for ∼40 min at 37°C in PSS1 containing 1 mg/mL collagenase type 2. After the incubation, the aorta was washed three times with PSS1 and the adventitia was carefully stripped. The aorta was cut longitudinally in order to expose the tunica intima and the endothelium was mechanically removed by rubbing a microdissection scissor against the endothelial layer. Aortic tissue was cut in a proximately 2 mm pieces and enzymatically digested for 40 min at 37°C in PSS1 containing 2 mg/mL collagenase type XI, 0.16 mg/mL elastase type IV and 2 mg/mL bovine serum albumin (BSA; fat free). After de digestion, VSMCs were resuspended by gently triturating the tissue with a fire-polished Pasteur pipette. To stop enzymatic digestion, 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) was added. VSMCs were centrifugated for 10 min at 300g. The supernatant was discarded, and the resulting cell pellet was re-suspended in 1 mL complete medium (DMEM with 10% FBS and 1% antibiotic antimycotic solution (100x)). VSMC were plated on 35 mm culture dishes under a humidified atmosphere of 5% CO₂ - 95% O₂ at 37°C. Culture medium was changed on days 4 and 7 and Ca²⁺ imaging experiments were performed on subconfluent cultures on days 7 and 8. The aortic VSMCs identity was confirmed by α-actin labelling, as shown in (Berra-Romani et al., 2008).

Cultured VSMCs were loaded with 3 µM Ca²⁺ indicator, Fura-2/AM in PSS2 (see composition in Section 2.5) for 35 min at controlled room temperature (22–23 °C). After 20 min washing in PSS2, the culture dish was mounted onto the stage of an upright epifluorescence Axiolab microscope (Carl Zeiss, Oberkochen, Germany) equipped with a ×40 Achroplan objective (water-immersion, 2.05 mm working distance, 1.0 numerical aperture) to observe the cells. VSMCs were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. A neutral density filter (optical density = 1.0) was coupled to the 380 nm filter to approach the intensity of the 340 nm light. A round diaphragm was used to increase the contrast. The exciting filters were mounted on a filter wheel equipped with a shutter (Lambda 10, Sutter Instrument, Novato, CA, United States). Custom software, working in the LINUX environment, was used to drive the camera (Extended-ISIS Camera, Photonic Science, Millham, United Kingdom) and the filter wheel, and to measure and plot on-line the fluorescence from a number of 20–50 rectangular “regions of interest” (ROI) enclosing 20–50 single cells. Each ROI was identified by a number. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed “Ratio (F₃₄₀/F₃₈₀)”. An increase in [Ca²⁺]_i causes an increase in the Ratio (F₃₄₀/F₃₈₀). Ratio measurements were performed and plotted on-line every 3 s. Ratio (F₃₄₀/F₃₈₀) values are expressed as arbitrary units (a.u.). Images were stored on the hard disk and converted offline to Ratio (F₃₄₀/F₃₈₀) images by ImageJ software (National Institutes of Health, United States, http://rsbweb.nih.gov/ij/). The experiments were carried out at controlled room temperature (22–23°C) to limit time-dependent decreases in the intensity of the fluorescence signal.

Mn²⁺ has been shown to quench Fura-2 fluorescence. Since Mn²⁺ and Ca²⁺ share common entry pathways in the plasma membrane, Fura-2 quenching by Mn²⁺ is regarded as an index of divalent cation influx. Experiments were carried out at the 360 nm wavelength, the isosbestic wavelength for Fura-2, and in Ca²⁺-free medium supplemented with 0.5 mM EGTA, as previously described (Zuccolo et al., 2019). This avoids Ca²⁺ competition for Mn²⁺ entry and, therefore, enhances Mn²⁺ quenching.

Low-Ca²⁺ physiological salt solution (PSS1) had the following composition (in mM): 140 NaCl, 5.36 KCl, 0.34 Na₂HPO₄, 0.44 KH₂PO₄, 10 HEPES, 1.2 MgCl₂, 10 D-glucose and 0.05 CaCl₂. Physiological salt solution (PSS2) composition (in mM) was: 140 NaCl, 5 KCl, 1.2 NaHPO₄, 5 NaHCO₃, 10 HEPES, 1.4 MgCl₂, 1.8 CaCl₂ and 11.5 D-glucose. In Ca²⁺ free solution (0Ca²⁺), 0.05 EGTA was added. In Mn²⁺-quenching experiments, 200 µM MnCl₂ was added to the 0Ca²⁺ external solution. Solutions were titrated to pH 7.2 for PSS1 and 7.4 for PSS2 with NaOH. The osmolality of PSS as measured with an osmometer (Wescor 5,500, Logan, UT) was 338 mmol/kg.

For each protocol, data were collected from at least four rats under each condition. The amplitude of the peak Ca²⁺ response to either cyclopiazonic acid (CPA) or adenosine-trisphosphate (ATP) was measured as the difference between the Ratio (F₃₄₀/F₃₈₀) at the peak and the mean Ratio (F₃₄₀/F₃₈₀) of 500 s baseline before the peak (Supplementary Figure S1). Amplitude of the late stage of the decay (Amp600) was calculated as the difference between the Ratio (F₃₄₀/F₃₈₀) 600 s after adding the agonist and the mean Ratio (F₃₄₀/F₃₈₀) of 200 s baseline before the peak of the Ca²⁺ response (Supplementary Figure S1). The duration of the Ca²⁺ response to ATP was measured as the time it takes the Ca²⁺ signal to be reduced at 90% (early) (Supplementary Figure S1), 60% (intermediate) (Supplementary Figure S1) and 30% (late) (Supplementary Figure S1) of the initial Ca²⁺ peak amplitude (considered as 100%), shorty termed as “decay time” (DT) (Supplementary Figure S1). The area under the curve (AUC) was measured by calculating the integral of each Ca²⁺ tracing from when the ATP is applied until it is removed. In order to perform the statistical comparison between the experimental groups in the presence of selective inhibitors of Ca²⁺- clearing mechanisms, we proceeded to normalize the measures to the mean value of each parameter determined in the absence of the inhibitor within the same experimental group (shortly termed “Δ”). For mean traces, fluorescence levels (F) were normalized to resting fluorescence (F₀) to compare the height of the Ca²⁺ responses produced by cells displaying different basal fluorescence levels (F/F₀). The rate of Mn²⁺ influx was evaluated by measuring the slope of the fluorescence intensity curve after Mn²⁺ addition (Zuccolo et al., 2019). Data are expressed as mean ± standard error (SE). Normal data (identified using the D’Agostino and Pearson omnibus normality test (p < 0.05)), were statistically analyzed using an unpaired Student’s t-test. A p-value < 0.05 was considered statistically significant.

SEA0400 was obtained from Tocris Bioscience (Bristol, United Kindom). Collagenase type 2 and FBS were obtained from Gibco (GIBCO BRL, Life Technologies, Grand Island, NY). All other chemicals were purchased from Sigma-Aldrich.

Somatic parameter obtained from 3 months old littermate LZDF and OZDF rats are shown in Table 1. OZDF rats presented an increase of around 66% in body mass weight (p ≤ 0.05) and of around 6.5% of the nose-tail length (p ≤ 0.05) compared with their control, LZDF rats. Given that the increase in weight could be attributed to an increase in the rat size (to its length), we proceeded to calculate the body mass index (BMI) to rule out that the differences seen in the weight were due to the difference in length between both groups. Likewise, the mean BMI value was statistically higher (46.6%) in the OZDF rats compared to the LZDF group (p ≤ 0.05), suggesting that the weight gain is due to obesity that has been widely reported in the literature in this strain of rats (Shiota and Printz, 2012; Wang et al., 2013; Al-Awar et al., 2016; King and Bowe, 2016; Berra-Romani et al., 2019). The presence of obesity in the OZDF rats was demonstrated by the 28% and 405% increase in abdominal circumference (p ≤ 0.05) and epididymal fat weight (p ≤ 0.05), respectively, in OZDF vs. LZDF rats. To confirm T2DM presence in OZDF rats, a glucose tolerance test was performed as described in Materials and Methods section. The mean ± SE of blood glucose levels measured 2 h after the intraperitoneal administration of glucose (2 g/kg of weight), was approximately 90% higher (p ≤ 0.05) in the OZDF rats compared to the LZDF rats (Table 1). Altogether the results confirm that OZDF rats present obesity and diabetes. In addition, a deeper biochemical analysis of LZDF rats and OZDF rats has been presented in (Berra-Romani et al., 2019), showing that OZDF rats present: 1) hyperlipidemia (increased total cholesterol, very low-density lipoproteins, and triglyceride levels); 2) hyperinsulinemia both in fasting and glucose-fed animals; and 3) insulin resistance. All together these results confirm the presence in OZDF rats of the main characteristics of human T2DM.

Preliminary recordings revealed that resting Ca²⁺ levels were similar (Supplementary Figure S2A) and that spontaneous Ca²⁺ oscillations (Supplementary Figure S2B) did not occur in VSMCs from the two animal groups. Moreover, 20 µM ATP was found to induce only a Ca²⁺ transient in rat aortic VSMCs from both OZDF and LZDF rats (Supplementary Figure S3). Since we were also interested in the impact of T2DM on the plateau phase of the Ca²⁺ response to physiological autacoids (Berra-Romani et al., 2019), ATP concentration was raised to 300 µM. In resting VSMCs from both LZDF and OZDF rats, the application of 300 µM ATP induced an intracellular Ca²⁺ signal that consisted in a rapid Ca²⁺ spike followed by a slow decay phase to a sustained plateau phase, which returned to the baseline upon agonist removal (Figure 1A). When comparing both groups, we did not find any statistical difference in both the peak amplitude (Figure 1B, left panel) and area under the curve (AUC) (Figure 1D). However, decay time (DT) at 90% and 60% of total amplitude were decreased (p ≤ 0.05) in VSMCs from OZDF rats (Figure 1C). In addition, we found an increase in the amplitude of the plateau phase in VSMCs from OZDF rats compared to LZDF rats, as shown by the increase in Amp600 value (p ≤ 0.05) (Figure 1B, right panel). Conversely, the decay time at 30% was not different between both groups (Figure 1C). The fastest decline of the initial Ca²⁺ peak could explain why the increase in Amp600 does not result in an increased AUC.

Figure 2 shows the average of Ca²⁺ signals recorded from cultured VSMCs obtained from LZDF (Figure 2A) and OZDF (Figure 2D) rat aortas challenged with ATP (300 μM) in presence (dotted line; + Ca²⁺) and absence (continuous line; + 0Ca²⁺) of Ca²⁺ in the extracellular medium. Removal of extracellular Ca²⁺ (0Ca²⁺) caused a decrease in peak amplitude, AUC, and decay time, as well as the disappearance of the plateau phase, in ATP-induced Ca²⁺ transients in both LZDF (Figures 2A-C) and OZDF VSMCs (Figures 2D–F). These findings confirm that both endogenous Ca²⁺ release through ER-resident IP₃Rs and extracellular Ca²⁺ entry via store-operated channels (SOCs) support the Ca²⁺ response to ATP in proliferating rat aortic VSMCs (Berra-Romani et al., 2008). In accord, depletion of the ER Ca²⁺ store with cyclopiazonic acid (CPA; 10 µM) prevented the subsequent Ca²⁺ response to ATP (300 µM) (Supplementary Figure S4). Of note, caffeine (10 mM), a selective agonist of ryanodine receptors (RyR) (Pulina et al., 2010) failed to elicit an increase in [Ca²⁺]_i in rat aortic VSMCs from LZDF rats (Supplementary Figure S3), which is consistent with the notion that proliferating VSMCs lack RyRs (Berra-Romani et al., 2008). Therefore, IP₃Rs drive ATP-dependent SR Ca²⁺ mobilization under 0Ca²⁺ conditions. Furthermore, the Mn²⁺-quenching assay, which has been widely employed to measure SOCE in both vascular endothelial cells (Zuccolo et al., 2019) and VSMCs (Smani et al., 2007), showed that CPA caused an increase in the rate of Fura-2 fluorescence quenching by extracellular Mn²⁺ (Supplementary Figure S5). However, the rate of CPA-evoked Mn²⁺ entry was significantly (p < 0.05) enhanced in rat aortic VSMCs from OZDF rats (Supplementary Figure S5), thereby confirming SOCE upregulation by T2DM.

In order to assess whether IP₃-induced SR Ca²⁺ release is affected by T2DM, we compared intracellular Ca²⁺ release evoked by the IP₃-synthesizing autacoid, ATP (300 µM), in the absence of extracellular Ca²⁺ (0Ca²⁺) in rat aortic VSMCs from LZDF and OZDF rats (Figure 3A) (Berra-Romani et al., 2008; Berra-Romani et al., 2019). Under these conditions, we found a reduction in both the peak Ca²⁺ amplitude and AUC of the Ca²⁺ transient in OZDF as compared to LZDF VSMCs (Figure 3B). Of note, the peak Ca²⁺ response to ATP was lower in rat aortic OZDF VSMCs in the absence, but not in the presence, of extracellular Ca²⁺ (see Figure 1B). We did not find any statistical difference in the decay time to 90% and 60% of the total amplitude; however, the decay time to 30% of the Ca²⁺ response was significantly (p < 0.05) increased in VSMCs from OZDF rats (Figure 3C). Conversely, in the presence of extracellular Ca²⁺, the early phase of the decay was faster in OZDF VSMCs and there was no difference in the decay time at 30% between the two animal groups (see Figure 1C). These observations suggest that: 1) endogenous Ca²⁺ release is reduced and/or 2) the Ca²⁺-transporting mechanisms clearing the initial Ca²⁺ peak are altered by T2DM, although this dysregulation is unmasked by removing extracellular Ca²⁺. The fastest decline of the initial Ca²⁺ peak recorded in the presence of external Ca²⁺ (Figure 1C) suggests that extracellular Ca²⁺ entry could stimulate SERCA, PMCA and/or NCX, as previously demonstrated in rat aortic VSMCs (Baryshnikov et al., 2009), and thereby accelerate cytosolic Ca²⁺ clearance (see Discussion). In addition, enhanced SOCE is also likely to compensate for the lower IP₃-induced SR Ca²⁺ release and bring the amplitude of the initial Ca²⁺ peak to the same value as that measured in LZDF rats.

The lower ER Ca²⁺ release evoked by the physiological stimulation of IP₃Rs with ATP could be due to a reduction in the free Ca²⁺ concentration within the ER. Therefore, we evaluated the amount of releasable free Ca²⁺ in the SR by inhibiting SERCA activity with CPA in the absence of extracellular Ca²⁺ (Berra-Romani et al., 2008; Berra-Romani et al., 2019). The application of CPA (10 µM) induced a slow Ca²⁺ transient corresponding to the passive leak of free SR Ca²⁺ into the cytoplasm through leakage channels that remain to be identified (Figure 4A). Subsequently, the intracellular Ca²⁺ concentration returned to the baseline because of Ca²⁺ extrusion across the sarcolemma by PMCA and NCX. CPA is routinely employed to compare SR or endoplasmic reticulum Ca²⁺ content between different cell types (Lodola et al., 2017; Berra-Romani et al., 2019), including VSMCs (Berra-Romani et al., 2008; Liu et al., 2019). No differences were found in the peak Ca²⁺ amplitude and AUC of the Ca²⁺ response to CPA (Figure 4B). These data indicate that there is no detectable difference in the SR free Ca²⁺ concentration between LZDF and OZDF VSMCs. Overall, these findings suggest that IP₃-induced SR Ca²⁺ release is somehow tempered in rat aortic VSMCs from OZDF rats and that there are no major differences in PMCA and NCX activity under 0Ca²⁺ conditions between the two animal groups (Figures 4A, B).

In order to assess whether the Ca²⁺-transporting mechanisms clearing the initial Ca²⁺ peak are altered by T2DM, we separately evaluated the contribution of SERCA, PMCA and NCX to ATP-induced increase in [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺. In accord, as anticipated above, extracellular Ca²⁺ entry could change the rate of cytosolic Ca²⁺ clearance from the cytosol. Therefore, the Ca²⁺-removal mechanisms at play during the physiological stimulation with ATP could differ depending on whether Ca²⁺ influx is activated or not.

We first evaluated whether and how T2DM alters SERCA activity by measuring the Ca²⁺ response to ATP (300 µM) in the presence of a selective SERCA inhibitor, CPA (10 µM).

To evaluate PMCA activity in rat aortic VSMCs during T2DM, we used orthovanadate (OV; 500 µM) to inhibit this mechanism during the stimulation with ATP in the presence and absence of extracellular Ca²⁺.

Finally, in order to assess whether and how T2DM alters NCX activity in rat aortic VSMCs, we inhibited this mechanism using the selective inhibitor, SEA0400 (3 µM), in the presence and absence of extracellular Ca²⁺ (0Ca²⁺).

As shown in Table 1, at the age of 12 weeks, all the morphological parameters measured (body weight, length, abdominal circumference and epididymal fat) were significantly increased in the OZDF rats in comparison to the LZDF group. The increment in weight concurs with the data previously reported by other research groups (Fridlyand and Philipson, 2006; Tirmenstein et al., 2015; Berra-Romani et al., 2019). Since this could be attributed to an increase in the length of the rat, we calculated the BMI to rule out the possibility that differences in length would interfere with our results. BMI was statistically higher in OZDF rats compared to the LZDF group, evidencing that weight gain is indeed due to obesity, as also reported by other studies (Shiota and Printz, 2012; Wang et al., 2013; Al-Awar et al., 2016; King and Bowe, 2016). The presence of obesity in OZDF rats was also demonstrated by a 405% increase in the amount of adipose tissue formed around the epididymis (epididymal fat). These results confirm that OZDF rats present obesity at the age of 12–16 weeks. Confirming that the animals used for the experimental protocols did indeed develop T2DM, plasma glucose levels 2 hours after intraperitoneally glucose administration (2 g/kg of weight) were more elevated in OZDF rats. This information indicates that OZDF rats are glucose intolerant, which constitutes one of the criteria that according to the ADA, are sufficient to diagnose T2DM in humans (ElSayed et al., 2023).

The experiments carried out in this study demonstrate that Ca²⁺ handling is altered in VSMCs from OZDF rats. In particular, T2DM causes two important effects on the ATP-evoked biphasic elevation in [Ca²⁺]_i: 1) a significant reduction in decay time to 90% and 60% of the initial Ca²⁺ peak amplitude (early stage of the decay of [Ca²⁺]_i to resting levels) and 2) a significant increase in the amplitude of the plateau (late stage of the decay of [Ca²⁺]_i and SOCE activation). Shortening of the early phase of decay could be due to alterations in the activity of the Ca²⁺-clearing mechanisms, such as SERCA, PMCA and NCX.

The role of SERCA (Zhang et al., 2018; Pereira et al., 2022); PMCA (Pande et al., 2006; Baryshnikov et al., 2009) and NCX (Liu et al., 2016; Yang et al., 2020) in clearing cytosolic Ca²⁺ upon agonist stimulation in aortic VSMCs has extensively been reported. However, few studies are available above the effect of DM on the activity of these Ca²⁺-clearing mechanisms VSMCs. In A7r5 cells, hyperglycemia was reported to induce a reduction in SERCA expression and activity (Searls et al., 2010; Tong et al., 2010) and an increase in PMCA and NCX activity (Han et al., 2022). However, our data showing that the decay phase of the initial increase in [Ca²⁺]_i evoked by ATP is accelerated in OZDF rat aortic VSMCs suggests that T2DM increases the activity of the Ca²⁺-clearing machinery. As to the increase in plateau amplitude observed in OZDF rat aortic VSMCs, this could clearly be due to SOCE upregulation, as recently shown in (Ma et al., 2020; Zhu et al., 2021). A recent report confirmed that SOCE was enhanced in aortic VSMCs deriving from Zucker diabetic fatty rats due to upregulation of Orai1 protein (Yang et al., 2020). In accord, the Mn²⁺-quenching assay confirmed that extracellular Ca²⁺ entry evoked by the pharmacological depletion of the ER Ca²⁺ store with CPA was remarkably larger in OZDF VSMCs as compared to LZDF. However, the increase in plateau amplitude could also reflect a reduction in the activity of Ca²⁺-extruding mechanisms and/or an increase in Ca²⁺ entry through the reverse mode of NCX (Zhang et al., 2005; Pulina et al., 2010; Liu et al., 2016; Berra-Romani et al., 2019).

In order to elucidate the mechanism(s) whereby T2DM alters the Ca²⁺ response to ATP in rat aortic VSMCs, we first stimulated the cells under 0Ca²⁺ conditions. This condition is widely employed to assess the role played by extracellular Ca²⁺ in shaping peak amplitude, decay phase and plateau amplitude of the Ca²⁺ response to extracellular stimulation (Urena et al., 2007; Berra-Romani et al., 2008; Linde et al., 2012). Ca²⁺ removal from the extracellular solution caused a significant decrease in Ca²⁺ peak amplitude and abolished the plateau phase, thus converting the biphasic Ca²⁺ response to ATP in a transient elevation in [Ca²⁺]_i in VSMCs from both animal groups. However, ATP-induced SR Ca²⁺ release was remarkably lower in rat aortic VSMCs from OZDF rats. Enhanced SOCE could therefore shift the amplitude of the initial Ca²⁺ peak evoked by ATP in OZDF VSMCs to the same amplitude as that induced in LZDF cells, which present a larger IP₃-induced SR Ca²⁺ release but lower SOCE. The reduced Ca²⁺ peak observed under 0Ca²⁺ conditions can be attributed to multiple factors, including downregulation of P2Y receptors, reduced phospholipase C (PLC) activation, lower IP₃R and RyR expression and/or activity, and a decrease in SR Ca²⁺ content. However, CPA-induced intracellular Ca²⁺ release, which is a widely employed readout of SR Ca²⁺ content, was not altered in OZDF VSMCs, as also reported in VSMCs from diabetic mice (Velmurugan and White, 2012). In contrast, ATP-evoked intracellular Ca²⁺ release, which is mainly dependent on IP₃Rs (see below), was significantly reduced in OZDF rat aortic VSMCs. This finding suggests that either P2Y receptors are downregulated or their downstream signalling pathways, such as those culminating in PLC engagement and IP₃R activation, are compromised by T2DM. A study performed in systemic arterial smooth muscle cells deriving from rat models of T1DM, as well as in VSMCs exposed to high glucose, showed a reduction in IP₃R expression that attenuated vasopressin-induced Ca²⁺ response (Searls et al., 2010). Conversely, Velmurugan et al. found that, in aortic VSMCs from mice with T2DM, IP₃R excitability was augmented through the direct interaction with the anti-apoptotic Bcl-2 proteins (Velmurugan and White, 2012). The discrepancy in these results could be due to several factors, such as heterogeneity in the vascular district, animal model, type of DM and progress of the disease. However, the evidence that T2DM increases IP₃R activity in mouse VSMCs strongly suggests that IP₃-dependent Ca²⁺ release is also enhanced in rat VSMCs. On the other hand, although DM alters RyR function in contractile VSMCs (Fernandez-Velasco et al., 2014), dedifferentiation induces RyR downregulation in the proliferative phenotype of VSMCs (Berra-Romani et al., 2008; House et al., 2008). Consistently, our Ca²⁺ imaging recordings confirmed that caffeine did not induce Ca²⁺ signals in cultured rat aortic VSMCs. Therefore, RyRs are unlikely to contribute to ATP-induced endogenous Ca²⁺ mobilization in the VSMCs employed in the present investigation. Likewise, ionotropic P2X receptors are lost during the dedifferentiation of VSMCs and cannot contribute to the initial Ca²⁺ peak induced by ATP in the presence of extracellular Ca²⁺ (Erlinge et al., 1998). The evidence discussed below that PMCA inhibition reduces the amplitude of the initial Ca²⁺ peak and that PMCA is engaged by Ca²⁺ entry strongly suggest that PMCA activity is required to promote full IP₃R activation in T2DM.

The inhibition of SERCA activity with CPA in the presence of extracellular Ca²⁺ elongated the decay phase of the Ca²⁺ transient induced by ATP in both experimental groups. However, this effect was greater during the early phases of decay (i.e., 90% and 60%) in OZDF rats. This observation suggests that Ca²⁺ sequestration by SERCA is enhanced during the initial stage of the Ca²⁺ transient and affects the early phase of decay in OZDF VSMCs. Furthermore, SERCA inhibition by CPA caused a larger increase in Amp600, which is a readout of plateau amplitude, in LZDF as compared to OZDF rats. This finding indicates that SERCA activity could decrease during the later stages of the decay phase, which would result in less removal of Ca²⁺ during the plateau phase. Therefore, the larger Amp600 recorded in OZDV VSMCs could be underlain by the combination of enhanced SOCE and reduced SERCA activity. Both an increase and a decrease in SERCA activity in the presence of DM have been reported. Particularly, in dyslipidemic diabetic pigs, Hill et al. found an increase in Ca²⁺ buffering by SERCA2 in coronary artery VSMCs isolated from dyslipidemic diabetic pigs (Hill et al., 2003). Conversely, in rat aortic VSMCs harvested from two different models of T1DM, Searls et al. found a decrease in the expression of SERCA2 and SERCA3, thereby reducing Ca²⁺ and attenuating the Ca²⁺ response to vasopressin (Searls et al., 2010). Downregulation of SERCA was also observed in cultured rat VSMCs exposed to high glucose, although this did not produce any significant alteration in the Ca²⁺ response to phenylephrine (El-Najjar et al., 2017). Interestingly, the protein expression of SERCA2B, which represents the main SERCA isoform in proliferating VSMCs (Berra-Romani et al., 2008) can be significantly enhanced in the thoracic and abdominal aortas of OZDF rats (Berra-Romani et al., 2019). Conversely, SERCA2B activity can be negatively modulated by oxidative stress (Berra-Romani et al., 2019; Negri et al., 2021), which is dramatically enhanced in OZDF rats (Chinen et al., 2007). It has been recently proposed that an early increase in [Ca²⁺]_i can stimulate the production of reactive oxygen species (ROS) via the Ca²⁺-dependent recruitment of NADPH oxidase 5 (NOX5) (Negri et al., 2021). Additionally, ROS could be generated upon NOX2 activation by Gq-protein Coupled Receptors (Negri et al., 2021), such as purinergic P2Y receptors. Therefore, we hypothesize that the initial increase in [Ca²⁺]_i evoked by ATP is rapidly cleared by SERCA2B in rat aortic VSMCs from OZDF rats due to its increased expression as compared to LZDF animals. However, ATP stimulation could also result in NOX2 and/or NOX5 activation, thereby exacerbating the ongoing oxidative stress and inhibiting SERCA2B activity during the plateau phase. Overall, our findings suggest that SERCA activity is increased during the initial phase of the Ca²⁺ response to an IP₃-producing autacoid, such as ATP, but the decreases during the later stages of the increase in [Ca²⁺]_i in aortic VSMCs from OZDF rats.

The next Ca²⁺-clearing mechanism evaluated in the present investigation was PMCA. In both experimental groups, OV caused a larger increase in Amp600 in LZDF as compared to OZDF rats. This finding indicates that also PMCA activity is reduced during the plateau phase of the Ca²⁺ response to ATP in diabetic animals. It turns out that blocking Ca²⁺ extrusion via PMCA with OV causes a larger accumulation of cytosolic Ca²⁺ in LZDF as compared to OZDF VSMCs. It is, therefore, plausible to conclude that the increase in plateau amplitude observed in diabetic rat aortic VSMCs is also due to a decrease in PMCA activity. As to the early phase of the Ca²⁺ signal, the decay time to 90% and 60% of the initial Ca²⁺ peak were significantly increased in OZDF, but not in LZDF VSMCs. This finding suggests that Ca²⁺ extrusion through PMCA is increased by T2DM at the onset of ATP-evoked increase in [Ca²⁺]_i. Notably, OV reduced the Ca²⁺ peak amplitude in diabetic but not lean VSMCs. This finding suggests that enhanced PMCA activity is somehow required to propagate the rise in [Ca²⁺]_i following agonist stimulation, as described in human platelets (Jones et al., 2010). The increase in PMCA activation could be attributed to enhanced SOCE, which can engage PMCA with high spatio-temporal efficiency and thereby finely tune IP₃-evoked SR Ca²⁺ release during the rising phase of the Ca²⁺ signal.

Conversely, when PMCA was inhibited in the absence of extracellular Ca²⁺, the decay time at 90%, 60% and 30% were significantly reduced in both animal groups. This finding strongly suggests that PMCA plays a minor role in buffering the initial increase in [Ca²⁺]_i under 0Ca²⁺ conditions. Furthermore, the amplitude of the initial Ca²⁺ peak was significantly decreased also in LZDF rat aortic VSMCs, which means that extracellular Ca²⁺ entry can substitute PMCA to regulate IP₃-dependent SR Ca²⁺ release in lean animals. Interestingly, also CPA reduced the amplitude of the initial Ca²⁺ peak in LZDF VSMCs stimulated with ATP only under 0Ca²⁺ conditions, thereby indicating that SERCA interacts with PMCA to finely tune IP₃-induced SR Ca²⁺ mobilization in non-diabetic VSMCs in the absence of Ca²⁺ entry.

These results, therefore, set extracellular Ca²⁺ as a regulator of PMCA in rat aortic VSMCs. PMCA activity is likely to be increased during the early phase of Ca²⁺ response evoked by ATP in the presence of extracellular Ca²⁺ in OZDF rat aortic VSMCs, while it declines during the subsequent plateau phase. In contrast, PMCA does not seem to have a relevant role under 0Ca²⁺ conditions in both animal groups. A functional coupling between SOCE and PMCA has long been known. Extracellular Ca²⁺ incoming through Orai1 channels can stimulate adjacent PMCA in a calmodulin-dependent manner and promote Ca²⁺ extrusion across the plasma membrane (Klishin et al., 1998; Snitsarev and Taylor, 1999). PMCA might not be engaged under 0Ca²⁺ conditions, so that IP₃-released Ca²⁺ is removed by alternative Ca²⁺-clearing mechanisms that present a higher extrusion rate and therefore accelerate the decay phase of the Ca²⁺ transient in both animal groups. The subsequent inhibition of PMCA activity during the late phase of decay even in the presence of extracellular Ca²⁺ could again reflect the enhanced oxidative stress imposed to diabetic VSMCs during the development of the Ca²⁺ signal. In accord, it has been demonstrated that ROS inhibit PMCA activity and thereby favour the accumulation of cytosolic Ca²⁺ (Kim et al., 2018), which is consistent with the increase in plateau amplitude in OZDF VSMCs.

Scarce information is available regarding the mechanisms by which T2DM affects PMCA in VSMCs; however, our results resemble those reported by El-Najjar et al., who showed that PMCA activity in rat aortic VSMCs exposed to high extracellular glucose is enhanced due to the increase in PMCA4 expression (El-Najjar et al., 2017). Similar results were observed in VSMCs from coronary arteries of diabetic and dyslipidemic pigs (Hill et al., 2003). Nevertheless, neither study evaluated whether PMCA activity decreases during the Ca²⁺ response to an IP₃-producing autacoid.

The last Ca²⁺-clearing mechanism evaluated in the present investigation was NCX. As described for SERCA and PMCA, NCX inhibition elicited different effects in lean vs. diabetic rats. In the presence of extracellular Ca²⁺, SEA0400 reduced the amplitude of the initial Ca²⁺ peak in LZDF but not OZDF VSMCs. Therefore, NCX is likely to regulate IP₃-induced SR Ca²⁺ release in rat aortic VSMCs from lean animals. Furthermore, blocking NCX activity did not remarkably affect the decay phase, but increased Amp600, of the Ca²⁺ response evoked by ATP in LZDF rats. Conversely, SEA0400 slowed down all the decay time and inhibited the plateau phase in OZDF VSMCs. These findings indicate that: 1) NCX operates in the forward (i.e., Ca²⁺ out) mode during the late phase of the Ca²⁺ response to ATP in lean VSMCs and 2) NCX switches from the forward to the reverse-mode (i.e., Ca²⁺ in) during the decay phase of the Ca²⁺ response evoked by ATP in diabetic VSMCs. The Ca²⁺-clearing activity of NCX during the early phase of the ATP-evoked increase in [Ca²⁺]_i could contribute to explain its sharper decline in OZDF VSMCs. Conversely, under 0Ca²⁺ conditions, Ca²⁺ extrusion via NCX seems to be decreased in diabetic VSMCs. In accord, blocking NCX activity accelerated the decay rate, which means that NCX is unlikely to clear cytosolic Ca²⁺ in the absence of Ca²⁺ entry. On the other hand, SEA0400 slows down the clearing rate in LZDF VSMCs, which indicates that in lean animals NCX drives the recovery of the Ca²⁺ transient to the baseline only under 0Ca²⁺ conditions.

Five Similar to SERCA, it is still unclear how T2DM affects NCX activity in VSMCs. A study performed by El-najjar et al. showed that, in VSMCs exposed to high glucose, there were no alterations in NCX expression or activity (El-najjar et al., 2017). Conversely, NCX activity was enhanced in cardiomyocytes isolated from db/db obese type 2 diabetic mice (Pereira et al., 2006). Future studies are required to understand whether NCX and PMCA expression are altered in aortic VSMCs from OZDF rats. Conclusion.

Herein, we provided the first demonstration that the Ca²⁺-transporting machinery is deranged in aortic VSMCs from diabetic obese rats. The Ca²⁺ pumping activity of SERCA and PMCA is increased by T2DM during the early phase of the Ca²⁺ transient, whereas it is remarkably slowed down during the following plateau phase. Similarly, NCX contributes to buffer the initial increase in [Ca²⁺]_i by operating in the reverse-mode, but it then reverses into the Ca²⁺-entry mode during the plateau phase. Therefore, the larger plateau observed in rat aortic VSMCs from OZDF rats is not only due to SOCE upregulation, as shown by others and confirmed in the present study, but also to the lower Ca²⁺ extrusion via PMCA and SERCA and to the activation of the reverse-mode of NCX. This latter observation suggests that a Na⁺-permeable channel is recruited or enhances its activity in diabetic VSMCs challenged with ATP. The non-selective cation channel, TRP Canonical 6 (TRPC6), can be gated by diacylglycerol produced upon PLC activation and generates local cytosolic Na⁺ transients beneath the plasma membrane that drive NCX-mediated Ca²⁺ entry in purinergically stimulated rat aortic VSMCs (Poburko et al., 2007). Future work will have to assess whether TRPC6 activation is enhanced by T2DM and contributes to further elevate the [Ca²⁺]_i during the plateau phase. Preliminary evidence showed that TRPC6 protein was upregulated in caudal artery smooth muscle from Type 2 diabetic Goto-Kakizaki rats (Mita et al., 2010), whereas TRPC3 protein expression was increased in platelets of patients affected by T2DM (Zbidi et al., 2009).

Intriguingly, extracellular Ca²⁺ entry is required to engage PMCA and the forward mode of NCX during the decay phase. The functional coupling between Orai1, i.e., the pore-forming subunit of SOCs, and PMCA has been firmly established, while a straight-forward relationship between SOCE and NCX-mediated Ca²⁺ extrusion is yet to be reported. The faster decay of [Ca²⁺]_i observed in diabetic rat aortic VSMCs challenged with ATP in the absence of extracellular Ca²⁺ and upon inhibition of either PMCA or NCX activity suggests the involvement of an alternative Ca²⁺-clearing mechanism. Mitochondrial have been shown to rapidly buffer Ca²⁺ at high [Ca²⁺]_i in rat systemic arterial smooth muscle cells (McCarron and Muir, 1999; Kamishima et al., 2000). Although this issue remains highly controversial, the rate of mitochondrial Ca²⁺ uptake could be increased by T2DM (Belosludtsev et al., 2020). Intriguingly, the simultaneous blockade of SERCA, PMCA and NCX activity in the absence of extracellular Ca²⁺ results in a plateau-like elevation in [Ca²⁺]_i that presents a higher Amp600 in VSMCs from LZDF rats. This finding implies that an additional Ca²⁺-removal mechanism intervene to buffer, at least partially, the initial rise in[Ca²⁺]_I elicited by ATP in rat aortic VSMCs from OZDF rats. These alterations in the Ca²⁺ clearing machine in aortic VSMCs could exacerbate vascular dysfunction in diabetes. For instance, the aberrant increase in [Ca²⁺]_i observed could enhance VSMC proliferation and migration, thereby favoring atherosclerotic plaques and vascular calcification. In addition, cytosolic Ca²⁺ overload in VSMCs could exacerbate oxidative stress within the vascular wall (Negri et al., 2021).