Genetic Ablation of Ca_V3.2 Channels Enhances the Arterial Myogenic Response by Modulating the RyR-BK_Ca Axis

Abstract

Objective

In resistance arteries, there is an emerging view that smooth muscle Ca_V3.2 channels restrain arterial constriction through a feedback response involving the large-conductance Ca²⁺-activated K⁺ channel (BK_Ca). Here, we used wild-type and Ca_V3.2 knockout (Ca_V3.2^−/−) mice to definitively test whether Ca_V3.2 moderates myogenic tone in mesenteric arteries via the Ca_V3.2-ryanodine receptor-BK_Ca axis and whether this regulatory mechanism influences blood pressure regulation.

Approach and Results

Using pressurized vessel myography, Ca_V3.2^−/− mesenteric arteries displayed enhanced myogenic constriction to pressure but similar K⁺-induced vasoconstriction compared with wild-type C57BL/6 arteries. Electrophysiological and myography experiments subsequently confirmed the inability of micromolar Ni²⁺, a Ca_V3.2 blocker, to either constrict arteries or suppress T-type currents in Ca_V3.2^−/− smooth muscle cells. The frequency of BK_Ca-induced spontaneous transient outward K⁺ currents dropped in wild-type but not in knockout arterial smooth muscle cells upon the pharmacological suppression of Ca_V3.2 channel. Line scan analysis performed on en face arteries loaded with Fluo-4 revealed the presence of Ca²⁺ sparks in all arteries, with the subsequent application of Ni²⁺ only affecting wild-type arteries. Although Ca_V3.2 channel moderated myogenic constriction of resistance arteries, the blood pressure measurements of Ca_V3.2^−/− and wild-type animals were similar.

Conclusions

Overall, our findings establish a negative feedback mechanism of the myogenic response in which Ca_V3.2 channel modulates downstream ryanodine receptor-BK_Ca to hyperpolarize and relax arteries.

The cardiovascular system comprises a muscular pump and a distribution network of arteries, veins, and capillaries. Within this integrated system, resistance arteries control the magnitude and distribution of tissue perfusion and respond to vasoactive stimuli, including mechanical forces, neurotransmitters, and metabolites.^1–5 Bayliss first described the inherent ability of resistance arteries to constrict to elevated pressure,⁶ and studies have shown that the so-called myogenic response is intimately tied to depolarization and the activation of smooth muscle L-type Ca²⁺ channels. It is often presumed that Ca_V1.2 is the only Ca²⁺ channel of functional significance because dihydropyridines, L-type blockers, prominently attenuate myogenic tone.³ This traditional perspective has begun to change with the identification of arterial T-type Ca²⁺ channels, including Ca_V3.1 and Ca_V3.2 subtypes.^7,8 Recent findings suggest that the former (ie, Ca_V3.1) modestly facilitates myogenic constriction at hyperpolarized voltages, whereas the latter (ie, Ca_V3.2) facilitates a negative feedback response restraining arterial constriction.^9,10

Our recent observations have tied the paradoxical ability of rat cerebral arterial Ca_V3.2 channel to limit myogenic tone to the triggering of ryanodine receptors (RyR) on the sarcoplasmic reticulum. The RyR-mediated generation of Ca²⁺ sparks subsequently activates the large conductance Ca²⁺-activated K⁺ channels (BK_Ca), eliciting a hyperpolarization to counteract pressure-induced constriction.¹⁰ Furthermore, the Ca_V3.2 conductance in the human cerebral circulation seems to mediate a similar physiological role.¹⁰ Although the concept of a voltage-gated Ca²⁺ channel counterbalancing vasoconstriction is novel and intriguing, it is one delimited by 2 primary concerns. First, current work is heavily reliant on the presumed selectivity of Ni²⁺ to block Ca_V3.2 channels.¹¹ Second, there is a lack of corroborative observations, outside the cerebral circulation, in vascular beds known to acutely and sustainably regulate systemic blood pressure.

Here, we used wild-type and Ca_V3.2 knockout (Ca_V3.2^−/−) mice to definitively test whether Ca_V3.2 channel moderates myogenic tone in mesenteric arteries via the Ca_V3.2-RyR-BK_Ca axis and, more generally, whether this regulatory mechanism influences blood pressure regulation. Experiments ranged from cells to whole animals and encompassed the integrative use of myography, electrophysiology, Ca²⁺ imaging, and intravascular catheterization. Arteries displayed enhanced myogenic tone when Ca_V3.2 channels were genetically ablated or pharmacologically suppressed using Ni²⁺. Subsequent analyses indicated that Ni²⁺ inhibited BK_Ca currents and Ca²⁺ sparks in wild-type but not Ca_V3.2^−/− arteries. Although Ca_V3.2 channel moderated myogenic constriction, the blood pressure measurements of both animal types were similar. In conclusion, this study establishes a negative feedback response in which Ca_V3.2 channel modulates downstream activity of the RyR-BK_Ca complex to hyperpolarize and relax resistance arteries.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

Genetic Ablation of Ca_V3.2 Enhances Arterial Myogenic Tone

Our earlier reports revealed the involvement of Ca_V3.2 channels in negative feedback control of rat cerebral arterial tone¹⁰; thus, channel deletion should enhance myogenic tone. Figure 1A and 1B demonstrates that C57BL/6 and Ca_V3.2^−/− arteries were myogenically active and constricted as intravascular pressure increased from 20 to 100 mm Hg. Ca²⁺-free saline (0 mmol/L Ca²⁺+2 mmol/L EGTA) reversed myogenic constriction and evoked passive arterial dilation. Expressing data as a percentage myogenic tone (Figure 1C) revealed the predicted enhancement in Ca_V3.2^−/− arteries (at 60 mm Hg: Ca_V3.2^−/−, 28%±2%; C57BL/6, 19%±3%). Although myogenic tone was significantly different, 60 mmol/L K⁺-induced vasoconstriction was similar in C57BL/6 (40%±2%) and Ca_V3.2^−/− (43%±2%) pressurized arteries (Figure 1D). Note, the basal diameter of Ca_V3.2^−/− arteries was smaller than that of C57BL/6, a finding consistent with variable myogenic tone. Interpretational cautious is, however, warranted because Ca_V3.2^−/− mice display a lighter body mass than wild-type animals.¹² Quantitative PCR showed that mRNA expression of Ca_V1.2, Ca_V3.1, BK_Ca-α, or RyR2 was comparable in wild-type and Ca_V3.2^−/− arteries (Figure 1E).

Figure 1.

Ca_V3.2^−/− arteries display enhanced myogenic tone. A, Representative traces and summary data demonstrate that C57BL/6 mesenteric arteries constricted to elevations in intravascular pressure from 20 to 100 mm Hg in Ca²⁺ containing physiological saline solution (PSS). Maximum arterial diameters were obtained by replacing Ca²⁺ PSS with PSS with zero Ca²⁺+2 mmol/L EGTA (n=12). B, Traces and averaged data of the myogenic response in Ca_V3.2^−/− mesenteric arteries (n=8). C, Percentage myogenic tone was significantly higher in Ca_V3.2^−/− (n=7) than in wild-type C57BL/6 (n=11) arteries (unpaired t test, *P≤0.05). D, Vasoconstriction evoked by 60 mmol/L K⁺ was similar in C57BL/6 and Ca_V3.2^−/− pressurized (15 mm Hg) arteries (n=11–17, unpaired t test; NS denotes not significant). E, Messenger RNA of key genes (Ca_V1.2, Ca_V3.1, BK_Ca-α, and RyR2) were not different in C57BL/6 and Ca_V3.2^−/− mesenteric arteries. Relative expression was calculated using 3 independent quantitative PCR reactions.

Micromolar Ni²⁺ Selectively Blocks Ca_V3.2 Currents

Mesenteric arterial smooth muscle cells from C57BL/6 mice express 3 subtypes of voltage-gated Ca²⁺ channels (Ca_V1.2, Ca_V3.1, and Ca_V3.2)⁹, and thus, total inward current is representative of this ensemble of channels. To distinguish between the subcomponents, we used patch clamp electrophysiology in combination with defined pharmacology.¹⁰ First, nifedipine (200 nmol/L) was applied to block L-type Ca_V1.2 channels and reveal a current predominated by T-type conductances (Figure 2A). Subsequent application of 50 μmol/L Ni²⁺, which is presumed to be a selective Ca_V3.2 blocker,^10–14 reduced the nifedipine-insensitive T-type currents in C57BL/6 but not in Ca_V3.2^−/− smooth muscle cells (Figure 2B and 2C). The absence of an effect of Ni²⁺ in Ca_V3.2^−/− myocytes is consistent with this pharmacological selectivity. Note that the broad-spectrum T-type blocker (NNC 55–0396, 1 μmol/L) subsequently abolished the residual current¹⁵ in both C57BL/6 and Ca_V3.2^−/− myocytes because of the suppression of remaining Ca_V3.1 current (Figure 2C). Importantly, voltage dependence profiles demonstrated that the T-type current is available for activation at physiological membrane potentials (Figure I in the online-only Data Supplement). As noted previously, Ca_V3.1 mRNA levels were similar among the 2 groups of animals, whereas Ca_V1.2 was modestly but insignificantly lower in Ca_V3.2^−/− arteries (Figure 1E).

Figure 2.

Micromolar Ni²⁺ suppresses Ca_V3.2 current. A, Current–voltage (I-V) plots illustrate total inward currents in wild-type C57BL/6 and Ca_V3.2^−/− smooth muscle cells and the effects of Ca_V1.2 and Ca_V3.2 blockade using nifedipine (200 nmol/L) and Ni²⁺ (50 μmol/L), respectively. All recordings used 10 mmol/L Ba²⁺ as the charge carrier. B and C, Representative traces and summary data illustrate the effect of Ni²⁺ on nifedipine insensitive T-type currents. Representative traces were evoked using a prepulse (−90 mV) followed by a test pulse (0 mV). Ni²⁺ significantly suppressed T-type currents in C57BL/6 but not Ca_V3.2^−/− smooth muscle cells (n=7 each; *P≤0.05).

Ca_V3.2 Activity Regulates BK_Ca-Mediated STOCs

Given the enhancement of myogenic constriction in Ca_V3.2^−/− arteries, we next tested whether this T-type channel modifies myogenic reactivity through a negative feedback response that involves downstream BK_Ca.^10,12 We used perforated patch clamp electrophysiology to monitor BK_Ca-mediated spontaneous transient outward K⁺ currents (STOCs) in arterial smooth muscle cells from wild-type and knockout animals. In C57BL/6 cells held at the physiological voltage of −40 mV, Ni²⁺ significantly suppressed STOC frequency. In contrast, Ni²⁺ had no effect on STOCs when C57BL/6 cells were voltage-clamped at more depolarized potentials (−20 mV), a finding consistent with the voltage profile of Ca_V3.2 channel (Figure 3A). In Ca_V3.2^−/− cells, Ni²⁺ had no effect at either −40 or −20 mV (Figure 3B). All STOCs were fully abolished by the application of the BK_Ca inhibitor paxilline (1 μmol/L;Figure II in the online-only Data Supplement). Message expression of the BK_Ca pore-forming subunit (BK_Ca-α) was similar in C57BL/6 and Ca_V3.2^−/− arteries (Figure 1E). Further, Ni²⁺ had no effect on STOC amplitude under different experimental conditions (Figure 3C). Note, basal STOCs tended to fire at lower frequencies in Ca_V3.2^−/− myocytes compared with C57BL/6 counterparts. Statistical analysis was not performed across groups as cells that did not fire sufficient STOCs were eliminated a priori from experimentation.

Figure 3.

Ca_V3.2 channel modulates BK_Ca-mediated spontaneous transient outward K⁺ currents (STOCs). A and B, Representative traces of STOCs recorded at −40 and −20 mV in wild-type C57BL/6 (A) or Ca_V3.2^−/− (B) arterial smooth muscle cells. Application of 50 μmol/L Ni²⁺ suppressed STOC frequency only at −40 mV in C57BL/6 cells with no noticeable effect at other conditions. C, Averaged bar graphs illustrate the effect of Ni²⁺ on STOC frequency (Hz) and amplitude (pA) at −40 or −20 mV in C57BL6 or Ca_V3.2^−/− arterial myocytes (n=8–11, *P≤0.05, paired t test).

Ca_V3.2 Channel Controls Ca²⁺ Spark Generation

Given the ability of Ca_V3.2 channel to modulate BK_Ca current (Figure 3) and the reported correlation between RyR-mediated spark generation and BK_Ca activation,^10,16 we next explored the Ca_V3.2-Ca²⁺ spark relationship using Ca²⁺ imaging and line scan analysis of mouse mesenteric arteries (Figure 4). In C57BL/6 arteries loaded with Fluo-4, Ca²⁺ sparks were observed in 76% of the 454 line scans performed under control conditions. Depolarizing arteries (30 mmol/L K⁺) increased spark activity (95%), whereas the subsequent addition of Ni²⁺ significantly reduced firing (52%; Figure 4A and 4B). In comparison, 30 mmol/L K⁺ increased spark firing in Ca_v3.2^−/− arteries from 58% to 94% (456 line scans) but Ni²⁺ failed to attenuate sparks (98%; Figure 4A and 4B). In depolarized wild-type C57BL/6 arteries, Ca²⁺ spark frequency was calculated to be 0.0347 and 0.0153 sparks/μm s in the absence and presence of Ni²⁺, respectively; spark frequency in knockout tissues was distinctively insensitive to the application of Ni²⁺ (Figure 4C). The genetic absence of Ca_V3.2 channels was notably associated with lower percentage firing (Ca_V3.2^−/−, 58%; C57BL/6, 76%) and lower basal Ca²⁺ spark frequency when compared with wild-type arteries (Ca_V3.2^−/−, 0.0065±0.0014; C57BL/6, 0.0169±0.005 sparks/μm s). The amplitudes and spatiotemporal characteristics of Ca²⁺ sparks displayed no significant changes before and after the application of Ni²⁺ on C57BL/6 and Ca_V3.2^−/− arteries (Figure 4D).

Figure 4.

Ca_V3.2 channel triggers Ca²⁺ spark generation. A, Representative line scan images (1.89 ms/line) of Fluo-4 loaded wild-type C57BL/6 or Ca_V3.2^−/− mesenteric arteries showing Ca²⁺ sparks recorded before and after the sequential application of 30 mmol/L K⁺ and 50 μmol/L Ni²⁺. The latter suppressed Ca²⁺ spark generation in C57BL/6 but not Ca_V3.2^−/− line scans. B, Percentage firing of Ca²⁺ sparks under control conditions and after the application of 30 mmol/L K⁺ followed by Ni²⁺. Line scans were recorded in C57BL/6 (n=454 line scans/9 animals) or Ca_V3.2^−/− (red, n=456 line scans/9 animals) mesenteric arteries (*P≤0.05, paired t test). C, Bar graph of Ca²⁺ spark frequency (sparks/μm s) before and after the application of Ni²⁺. Ca_V3.2 suppression significantly attenuated spark frequency in C57BL/6 (n=9, 454 line scans in total, *P≤0.05) but not Ca_V3.2^−/− (n=9, 456 line scans in total) arteries. D, Bar graphs of averaged Ca²⁺ spark amplitude (F/F₀); full width at half maximum (FWHM, in μm); full duration at half maximum (FDHM, in ms); and time constants of decay (Tau, in ms) before and after Ni²⁺.

Ca_V3.2 Activity Restrains Myogenic Constriction by Altering BK_Ca Feedback

The application of Ni²⁺ (50 μmol/L) onto C57BL/6 arteries evoked vasoconstriction at intravascular pressure values between 20 and 60 mm Hg, and this vasomotor effect diminished at higher pressures; Ca_V3.2^−/− arteries lacked a similar response (Figure 5A). The percentage of myogenic tone in wild-type C57BL/6 arteries increased after the application of Ni²⁺ (at 60 mm Hg: control 20%±4% versus Ni²⁺ 25%±1%), but was not altered in Ca_V3.2^−/− arteries (control 30%±5% versus Ni²⁺ 28%±8%; Figure 5B). Coinciding with vasomotor data, membrane potential measurements showed that Ni²⁺ only depolarized C57BL/6 but not Ca_V3.2^−/− pressurized arteries (60 mm Hg; Figure 5C). In C57BL/6 arteries, the BK_Ca blocker (paxilline, 1 μmol/L) evoked vasoconstriction and enhanced myogenic tone similar to that of Ni²⁺ (Figure 5D), an observation consistent with a common signaling axis between Ca_V3.2 and BK_Ca channels. When Ni²⁺ and paxilline were sequentially added to the same wild-type artery, Ni²⁺ evoked vasoconstriction, whereas subsequent paxilline had no additional effect. Similar experiments using Ca_V3.2^−/− arteries demonstrated a lack of vasomotor responses to Ni²⁺ but preserved responsiveness to paxilline (Figure 5E).

Figure 5.

Ca_V3.2 suppression constricts/depolarizes wild-type arteries via altering the vasomotor BK_Ca response. A, Averaged data demonstrate that the application of 50 μmol/L Ni²⁺ constricted pressurized C57BL/6 arteries (n=9) at pressure values 20 to 60 mm Hg with no significant effect on Ca_V3.2^−/− arteries (n=4). B, Ca_V3.2 blockade enhanced percentage myogenic tone in wild-type C57BL/6 arteries, but displayed no detectable change in Ca_V3.2^−/− arteries. C, Representative traces and summary data of membrane potential (mV) in C57BL/6 and Ca_V3.2^−/− pressurized arteries (60 mm Hg) before and after the application of Ni²⁺ (n=6 each; paired t test; *P≤0.05). D, Application of Ni²⁺ (50 μmol/L, n=9, as in A) or paxilline (1 μmol/L, n=4) on pressurized C57BL/6 arteries elicited similar constrictor responses. Percentage myogenic tone increased in response to BK_Ca blockade in C57BL/6 arteries (n=4). E, Changes in arterial diameter in response to Ni²⁺ followed by paxilline in pressurized C57BL/6 or Ca_V3.2^−/− arteries (at 60 mm Hg, n=5–7, *P≤0.05).

Ca_V3.2^−/− Mice Display Normal Blood Pressure Responses

Pharmacological and genetic approaches suggested that Ca_V3.2 channel counterbalances myogenic constriction (Figure 1 and 5) and could as such influence blood pressure regulation. To explore this possibility, we catheterized carotid arteries of C57BL/6 and Ca_V3.2^−/− mice to monitor blood pressure under resting conditions and in response to a vasopressor challenge. As depicted (Figure 6A), basal mean arterial pressure was similar in wild-type and knockout mice (C57BL/6, 100±2 mm Hg; Ca_V3.2^−/−, 103±5 mm Hg), a finding consistent with earlier reports in conscious animals.^17,18 Given that Ca_V3.2 channels seem to be involved in a feedback mechanism, we next assessed whether this conductance can alter mean arterial pressure responsiveness to a vasopressor challenge. The intravenous administration of phenylephrine (α₁-adrenoceptor agonist, 1–16 μg/kg body weight) evoked dose-dependent rises in mean arterial pressure, and these transient responses were similar among the 2 groups (Figure 6B). Analogous to in vivo experiments, phenylephrine (0.01–10 μmol/L)-induced vasoconstriction was similar in C57BL/6 and Ca_V3.2^−/− mesenteric arteries (Figure 6C and 6D).

Figure 6.

Ca_V3.2^−/− mice display normal resting blood pressure and responsiveness to phenylephrine. A, Resting mean arterial pressure of anesthetized C57BL/6 or Ca_V3.2^−/− mice (n=10 each). B, Changes in mean arterial pressure (ΔMAP, mm Hg) in response to intravenous phenylephrine (1–16 μg/kg body weight) in C57BL/6 and Ca_V3.2^−/− mice (n=10 each). C, Diameter responses of C57BL/6 and Ca_V3.2^−/− mesenteric arteries to increased pressure from 15 to 60 mm Hg followed by cumulative application of phenylephrine (PE; 0.01–10 μmol/L). Maximum diameters (D_max) were finally achieved by perfusion of Ca²⁺ free PSS. D, Phenylephrine concentration response curve shows no significant differences between wild-type C57BL/6 and Ca_V3.2^−/− arteries (n=8 each).

Discussion

This study used wild-type and Ca_V3.2 knockout mice to examine the purported contribution of Ca_V3.2 channels to a negative feedback response that counterbalances arterial tone development. Using mesenteric arteries, functional experiments illustrated that the genetic ablation or pharmacological suppression of Ca_V3.2 channel selectively enhanced myogenic constriction. Subsequent electrophysiological recordings revealed that Ca_V3.2 channel modulates downstream BK_Ca-mediated STOCs. Ca²⁺ imaging further demonstrated that Ca²⁺ spark generation is an intermediary step in the Ca_V3.2-BK_Ca functional axis. Finally, although Ca_V3.2 moderated myogenic tone, this regulatory mechanism did not influence resting blood pressure or vasopressor-induced responses. In summary, findings from this study establish a model by which Ca_V3.2 channel restrains myogenic constriction by driving a process where Ca²⁺ influx triggers Ca²⁺ sparks and downstream activation of BK_Ca currents (Figure III in the online-only Data Supplement).

Resistance arteries control tissue perfusion and respond to a range of vasoactive stimuli.^1–5 The integral ability of resistance arteries to respond to perturbations in arterial pressure, known as the myogenic response,⁶ is intrinsic to vascular smooth muscle and plays an essential role in maintaining blood flow and capillary pressure to tissues, such as the brain and the heart.¹⁹ This response is mechanistically linked to a rise in cytosolic [Ca²⁺]_i driven by a depolarization that activates L-type Ca_V1.2 channels. The traditional perspective that Ca_V1.2 is the only voltage-gated Ca²⁺ channel of functional significance³ has shifted with the identification of 2 T-type Ca²⁺ channels,^7,8 the first being Ca_V3.1, which modestly facilitates myogenic tone at hyperpolarized voltages.⁹ In contrast, Ca_V3.2 channels have been reported to drive a paradoxical feedback response that limits arterial constriction.^10,12 The feedback response begins with Ca²⁺ influx through Ca_V3.2 channels triggering ryanodine receptors to initiate Ca²⁺ sparks. These discrete sarcoplasmic reticulum–driven events in turn activate BK_Ca channels to hyperpolarize and dilate resistance arteries.¹⁰ Although an intriguing concept, data interpretation is singularly dependent on the presumed selectivity of Ni²⁺ as a pharmacological Ca_V3.2 blocker.¹¹ Current knowledge is also restricted to the cerebral circulation, and experiments have not extended to other vascular beds essential to blood pressure regulation.

One means to better probe the functional properties of arterial Ca_V3.2 channel is to use an animal model in which the channel has been genetically ablated.¹² In this regard, we performed pressure myography on arteries from Ca_V3.2 knockout (Ca_V3.2^−/−) mice to study the vascular phenotype. Consistent with the view that Ca_V3.2 channel mediates feedback vasodilation, we observed enhanced myogenic constriction in mesenteric arteries from Ca_V3.2^−/− mice. This finding was somewhat akin to the functional observations in coronary arteries¹² and the enhancement of rat cerebral arterial myogenic tone noted in the presence of Ni²⁺.¹⁰ Our present findings overcome concerns raised by past studies that off-target effects of Ni²⁺ could theoretically have accounted for the vasomotor responses.^20–22 In particular, we were unable to elicit Ni²⁺-sensitive current in Ca_V3.2^−/− arterial myocytes, and Ni²⁺ also failed to constrict/depolarize arteries from knockout mice.

The unexpected ability of Ca_V3.2 channel to mediate a negative feedback response has been previously tied to downstream modulation of BK_Ca channels through intermediary activation of ryanodine receptors.¹⁰ In arterial smooth muscle, BK_Ca is ubiquitously expressed and known to moderate membrane depolarization and arterial constriction.²³ To activate arterial BK_Ca, vasoactive stimuli must induce depolarization and elicit a discrete micromolar rise in [Ca²⁺]_i in the subsarcolemma taking the form of a Ca²⁺ spark.²⁴ Here, we present multiple lines of evidence implicating BK_Ca as the final downstream effector of Ca_V3.2 channel. First, electrophysiology revealed that BK_Ca-mediated STOCs were sensitive to Ni²⁺ in wild-type but not in Ca_V3.2^−/− arterial myocytes. Second, pressure myography illustrated that adding Ni²⁺ or paxilline (BK_Ca inhibitor) to the superfusate comparably augmented myogenic tone. Intriguingly, applying paxilline to wild-type arteries pre-treated with Ni²⁺ had no additive effect, consistent with Ca_V3.2 and BK_Ca channels being linked through a common sequential pathway.

It has been long established that RyR activation is responsible for the initiation of BK_Ca-mediated STOCs in arterial smooth muscle,^16,24 and this recognized relationship led us to examine the nature of Ca²⁺ spark generation in mouse mesenteric arteries. Indeed, inhibitors of RyR (eg, ryanodine) has been shown to suppress the generation of STOCs in vascular smooth muscle irrespective of their origin.²⁴ Here and in consistency with Ca_V3.2 channel driving Ca²⁺ sparks and subsequently STOC generation, we used Ca²⁺ imaging and line scan analysis and observed that spark frequency decreased in wild-type arteries when Ca_V3.2 channels were inhibited with Ni²⁺. We also found that firing of Ca²⁺ sparks was unaffected by this divalent cation in Ca_V3.2^−/− arteries and that basal Ca²⁺ spark frequency was lower than those of wild-type arteries. These findings align well with recent work from the cerebral circulation where a variety of functional, structural, electro-physiological, and computational observations draw a critical relationship between Ca_V3.2 and RyR and then BK_Ca (Figure III in the online-only Data Supplement).¹⁰ Intriguingly, the preceding work is distinct from neuronal studies where T-type conductances have been suggested to directly activate Ca²⁺-activated K⁺ channels independent of RyR.^25–28

With Ca_V3.2 channels playing an intimate role in limiting myogenic constriction, it is logical to argue that the loss of this conductance would alter peripheral resistance and systemic blood pressure regulation. We tested this supposition by catheterizing anesthetized mice and assessing blood pressure at rest and in response to a vasopressor challenge. Resting blood pressure was similar among wild-type and knockout mice, a finding consistent with earlier reports which used different monitoring approaches (eg, catheterization, tail cuff) in conscious animals over longer time spans (days to weeks).^17,18 Given the presumed role of Ca_V3.2 in feedback (rather than active vasodilation), we challenged blood pressure regulation using intravenous phenylephrine. This multidose challenge evoked comparable rises in blood pressure among the 2 animal groups; likewise, mesenteric arteries isolated from wild-type and Ca_V3.2^−/− mice constricted similarly to this α₁-adrenergic agonist. These in vivo and in vitro results interestingly seem to rule out a direct modulatory role for vascular Ca_V3.2 channels in agonist-induced vasoconstriction.

It is not clear at present why blood pressure responses fail to change. Perhaps, the loss of smooth muscle feedback in Ca_V3.2^−/− arteries is compensated by enhanced dilatory feedback from the endothelium. Other functional compensation may have occurred in this global knockout independent of vascular smooth muscle²⁹; in this context, establishing a smooth muscle–specific Ca_V3.2 knockout model would be valuable. It should also be recognized that for the proposed Ca_V3.2/RyR/BK_Ca feedback mechanism to effectively drive a blood pressure response, it should be ideally present in a full range of vascular beds. Recent work documenting heterogeneity of BK_Ca activity,³⁰ because of differences in channel activity and Ca²⁺ spark generation, challenges this key assumption. Further investigation is warranted to address this intriguing anomaly.

In conclusion, this study showed that arterial Ca_V3.2 channels retain a unique ability to counterbalance myogenic constriction. This negative feedback response entails a modulatory paradigm in which Ca²⁺ flux through Ca_V3.2 channel triggers Ca²⁺ spark generation and then activates BK_Ca channels to hyperpolarize and relax arteries (Figure III in the online-only Data Supplement). This novel functional axis, recently described in the human cerebral circulation,³¹ challenges the traditional view that voltage-gated Ca²⁺ channels solely facilitate arterial tone development.

Significance.

Vascular smooth muscle cells express T-type Ca²⁺ channels along with L-type channels. Although the latter have been long implicated in arterial excitation–contraction coupling, studies have only recently begun to assess the role of T-type channels. Using an animal model in which Ca_V3.2, a T-type channel, was genetically deleted, we tested its role in arterial tone development. We demonstrate that resistance arteries from knockout animals paradoxically display enhanced responsiveness to arterial pressure. This enhancement was mechanistically attributed to the ability of Ca_V3.2 to modulate downstream K⁺ channels, which hyperpolarize and relax arteries. This novel data challenge the traditional view that voltage-gated Ca²⁺ channels are singularly involved in the genesis of arterial constriction.

Nonstandard Abbreviations and Acronyms

BK_Ca

large-conductance Ca²⁺-activated K⁺ channel

Ca_V3.2^−/−

Ca_V3.2-deficient (knockout) mice

RyR

ryanodine receptor

STOC

spontaneous transient outward K⁺ current