CaV3.1 channels facilitate calcium wave generation and myogenic tone development in mouse mesenteric arteries.

The arterial myogenic response to intraluminal pressure elicits constriction to maintain tissue perfusion. Smooth muscle [Ca2+] is a key determinant of constriction, tied to L-type (CaV1.2) Ca2+ channels. While important, other Ca2+ channels, particularly T-type could contribute to pressure regulation within defined voltage ranges. This study examined the role of one T-type Ca2+ channel (CaV3.1) using C57BL/6 wild type and CaV3.1-/- mice. Patch-clamp electrophysiology, pressure myography, blood pressure and Ca2+ imaging defined the CaV3.1-/- phenotype relative to C57BL/6. CaV3.1-/- mice had absent CaV3.1 expression and whole-cell current, coinciding with lower blood pressure and reduced mesenteric artery myogenic tone, particularly at lower pressures (20-60 mmHg) where membrane potential is hyperpolarized. This reduction coincided with diminished Ca2+ wave generation, asynchronous events of Ca2+ release from the sarcoplasmic reticulum, insensitive to L-type Ca2+ channel blockade (Nifedipine, 0.3 µM). Proximity ligation assay (PLA) confirmed IP3R1/CaV3.1 close physical association. IP3R blockade (2-APB, 50 µM or xestospongin C, 3 µM) in nifedipine-treated C57BL/6 arteries rendered a CaV3.1-/- contractile phenotype. Findings indicate that Ca2+ influx through CaV3.1 contributes to myogenic tone at hyperpolarized voltages through Ca2+-induced Ca2+ release tied to the sarcoplasmic reticulum. This study helps establish CaV3.1 as a potential therapeutic target to control blood pressure.

Decision Making in Mice During an Optimized Touchscreen Spatial Working Memory Task Sensitive to Medial Prefrontal Cortex Inactivation and NMDA Receptor Hypofunction.

Working memory is a fundamental cognitive process for decision-making and is a hallmark impairment in a variety of neuropsychiatric and neurodegenerative diseases. Spatial working memory paradigms are a valuable tool to assess these processes in rodents and dissect the neurobiology underlying working memory. The trial unique non-match to location (TUNL) task is an automated touchscreen paradigm used to study spatial working memory and pattern separation processes in rodents. Here, animals must remember the spatial location of a stimulus presented on the screen over a delay period; and use this representation to respond to the novel location when the two are presented together. Because stimuli can be presented in a variety of spatial configurations, TUNL offers a trial-unique paradigm, which can aid in combating the development of unwanted mediating strategies. Here, we have optimized the TUNL protocol for mice to reduce training time and further reduce the potential development of mediating strategies. As a result, mice are able to accurately perform an enhanced trial-unique paradigm, where the locations of the sample and choice stimuli can be presented in any configuration on the screen during a single session. We also aimed to pharmacologically characterize this updated protocol, by assessing the roles of the medial prefrontal cortex (mPFC) and N-methyl-D-aspartate (NMDA) receptor (NMDAr) functioning during TUNL. Temporary inactivation of the medial prefrontal cortex (mPFC) was accomplished by directly infusing a mixture of GABA agonists muscimol and baclofen into the mPFC. We found that mPFC inactivation significantly impaired TUNL performance in a delay-dependent manner. In addition, mPFC inactivation significantly increased the susceptibility of mice to proactive interference. Mice were then challenged with acute systemic injections of the NMDAr antagonist ketamine, which resulted in a dose-dependent, delay-dependent working memory impairment. Together, we describe an optimized automated touchscreen task of working memory, which is dependent on the intact functioning of the mPFC and sensitive to acute NMDAr hypofunction. With the vast genetic toolbox available for modeling disease and probing neural circuit functioning in mice, the TUNL task offers a valuable paradigm to pair with these technologies to further investigate the processes underlying spatial working memory.

Defining a role of NADPH oxidase in myogenic tone development.

OBJECTIVE: The myogenic response sets the foundation for blood flow control. Recent findings suggest a role for G protein-coupled receptors (GPCR) and signaling pathways tied to the generation of reactive oxygen species (ROS). In this regard, this study ascertained the impact of NADPH oxidase (Nox) on myogenic tone in rat cerebral resistance arteries. METHODS: The study employed real-time qPCR (RT-qPCR), pressure myography, and immunohistochemistry. RESULTS: Gq blockade abolished myogenic tone in rat cerebral arteries, linking GPCR to mechanosensation. Subsequent work revealed that general (TEMPOL) and mitochondrial specific (MitoTEMPO) ROS scavengers had little impact on myogenic tone, whereas apocynin, a broad spectrum Nox inhibitor, initiated transient dilation. RT-qPCR revealed Nox1 and Nox2 mRNA expression in smooth muscle cells. Pressure myography defined Nox1 rather than Nox2 is facilitating myogenic tone. We rationalized that Nox1-generated ROS was initiating this response by impairing the ability of the CaV 3.2 channel to elicit negative feedback via BKCa . This hypothesis was confirmed in functional experiments. The proximity ligation assay further revealed that Nox1 and CaV 3.2 colocalize within 40 nm of one another. CONCLUSIONS: Our data highlight that vascular pressurization augments Nox1 activity and ensuing ROS production facilitates myogenic tone by limiting Ca2+ influx via CaV 3.2.

Caveolae Link CaV3.2 Channels to BKCa-Mediated Feedback in Vascular Smooth Muscle.

Objective- This study examined whether caveolae position CaV3.2 (T-type Ca2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large-conductance Ca2+-activated K+ channel feedback. Approach and Results- Using smooth muscle cells from mouse mesenteric arteries, the proximity ligation assay confirmed that CaV3.2 reside within 40 nm of caveolin 1, a key caveolae protein. Methyl-ß-cyclodextrin, a cholesterol depleting agent that disrupts caveolae, suppressed CaV3.2 activity along with large-conductance Ca2+-activated K+-mediated spontaneous transient outward currents in cells from C57BL/6 but not CaV3.2-/- mice. Genetic deletion of caveolin 1, a perturbation that prevents caveolae formation, also impaired spontaneous transient outward current production but did so without impairing Ca2+ channel activity, including CaV3.2. These observations indicate a mistargeting of CaV3.2 in caveolin 1-/- mice, a view supported by a loss of Ni2+-sensitive Ca2+ spark generation and colocalization signal (CaV3.2-RyR) from the proximity ligation assay. Vasomotor and membrane potential measurements confirmed that cellular disruption of the CaV3.2-RyR axis functionally impaired the ability of large-conductance Ca2+-activated K+ to set tone in pressurized caveolin 1-/- arteries. Conclusions- Caveolae play a critical role in protein targeting and preserving the close structural relationship between CaV3.2 and RyR needed to drive negative feedback control in resistance arteries.

Differential targeting and signalling of voltage-gated T-type Cav 3.2 and L-type Cav 1.2 channels to ryanodine receptors in mesenteric arteries.

KEY POINTS: In arterial smooth muscle, Ca2+ sparks are elementary Ca2+ -release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca2+ -sensitive K+ (BKCa ) channels. This study elucidated the contribution of T-type Cav 3.2 channels in caveolae and their functional interaction with L-type Cav 1.2 channels to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). Our data demonstrate that L-type Cav 1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs. T-type Cav 3.2 channels represent an additional source for generation of VSMC Ca2+ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T-type Cav 3.2 channels and RyRs to ignite Ca2+ sparks. ABSTRACT: Recent data suggest that T-type Cav 3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca2+ signalling (Ca2+ sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca2+ sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Cav 3.2 channels and their functional interaction with Cav 1.2 channels to trigger Ca2+ sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen-inducible smooth muscle-specific Cav 1.2-/- (SMAKO) mice and laser scanning confocal microscopy to assess Ca2+ spark generation in VSMCs. Ni2+ , Cd2+ and methyl-ß-cyclodextrin were used to inhibit Cav 3.2 channels, Cav 1.2 channels and caveolae, respectively. Ni2+ (50 µmol L-1 ) and methyl-ß-cyclodextrin (10 mmol L-1 ) decreased Ca2+ spark frequency by ∼20-30% in mesenteric VSMCs in a non-additive manner, but failed to inhibit Ca2+ sparks in tibial and cerebral artery VSMCs. Cd2+ (200 µmol L-1 ) suppressed Ca2+ sparks in mesenteric arteries by ∼70-80%. A similar suppression of Ca2+ sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca2+ sparks were fully abolished by Ni2+ or methyl-ß-cyclodextrin. Our data demonstrate that Ca2+ influx through CaV 1.2 channels is the primary means of triggering Ca2+ sparks in murine arterial VSMCs. CaV 3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca2+ source for Ca2+ spark generation in mesenteric, but not tibial and cerebral, arteries.

Reactive Oxygen Species Mediate the Suppression of Arterial Smooth Muscle T-type Ca2+ Channels by Angiotensin II.

Vascular T-type Ca2+ channels (CaV3.1 and CaV3.2) play a key role in arterial tone development. This study investigated whether this conductance is a regulatory target of angiotensin II (Ang II), a vasoactive peptide that circulates and which is locally produced within the arterial wall. Patch clamp electrophysiology performed on rat cerebral arterial smooth muscle cells reveals that Ang II (100 nM) inhibited T-type currents through AT1 receptor activation. Blocking protein kinase C failed to eliminate channel suppression, a finding consistent with unique signaling proteins enabling this response. In this regard, inhibiting NADPH oxidase (Nox) with apocynin or ML171 (Nox1 selective) abolished channel suppression highlighting a role for reactive oxygen species (ROS). In the presence of Ni2+ (50 µM), Ang II failed to modulate the residual T-type current, an observation consistent with this peptide targeting CaV3.2. Selective channel suppression by Ang II impaired the ability of CaV3.2 to alter spontaneous transient outward currents or vessel diameter. Proximity ligation assay confirmed Nox1 colocalization with CaV3.2. In closing, Ang II targets CaV3.2 channels via a signaling pathway involving Nox1 and the generation of ROS. This unique regulatory mechanism alters BKCa mediated feedback giving rise to a "constrictive" phenotype often observed with cerebrovascular disease.

Interplay among distinct Ca2+ conductances drives Ca2+ sparks/spontaneous transient outward currents in rat cerebral arteries.

KEY POINTS: Distinct Ca2+ channels work in a coordinated manner to grade Ca2+ spark/spontaneous transient outward currents (STOCs) in rat cerebral arteries. The relative contribution of each Ca2+ channel to Ca2+ spark/STOC production depends upon their biophysical properties and the resting membrane potential of smooth muscle. Na+ /Ca2+ exchanger, but not TRP channels, can also facilitate STOC production. ABSTRACT: Ca2+ sparks are generated in a voltage-dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction. In this study, we defined the mechanisms by which membrane depolarization increases Ca2+ sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOCs in the presence and absence of agents that modulate Ca2+ entry. Beginning with CaV 3.2 channel inhibition, Ni2+ was shown to decrease STOC frequency in cells held at hyperpolarized (-40 mV) but not depolarized (-20 mV) voltages. In contrast, nifedipine, a CaV 1.2 inhibitor, markedly suppressed STOC frequency at -20 mV but not -40 mV. These findings aligned with the voltage-dependent profiles of L- and T-type Ca2+ channels. Furthermore, computational and experimental observations illustrated that Ca2+ spark production is intimately tied to the activity of both conductances. Intriguingly, this study observed residual STOC production at depolarized voltages that was independent of CaV 1.2 and CaV 3.2. This residual component was insensitive to TRPV4 channel modulation and was abolished by Na+ /Ca2+ exchanger blockade. In summary, our work highlights that the voltage-dependent triggering of Ca2+ sparks/STOCs is not tied to a single conductance but rather reflects an interplay among multiple Ca2+ permeable pores with distinct electrophysiological properties. This integrated orchestration enables smooth muscle to grade Ca2+ spark/STOC production and thus precisely tune negative electrical feedback.

KIR channels tune electrical communication in cerebral arteries.

The conducted vasomotor response reflects electrical communication in the arterial wall and the distance signals spread is regulated by three factors including resident ion channels. This study defined the role of inward-rectifying K+ channels (KIR) in governing electrical communication along hamster cerebral arteries. Focal KCl application induced a vasoconstriction that conducted robustly, indicative of electrical communication among cells. Inhibiting dominant K+ conductances had no attenuating effect, the exception being Ba2+ blockade of KIR. Electrophysiology and Q-PCR analysis of smooth muscle cells revealed a Ba2+-sensitive KIR current comprised of KIR2.1/2.2 subunits. This current was surprisingly small and when incorporated into a model, failed to account for the observed changes in conduction. We theorized a second population of KIR channels exist and consistent with this idea, a robust Ba2+-sensitive KIR2.1/2.2 current was observed in endothelial cells. When both KIR currents were incorporated into, and then inhibited in our model, conduction decay was substantive, aligning with experiments. Enhanced decay was ascribed to the rightward shift in membrane potential and the increased feedback arising from voltage-dependent-K+ channels. In summary, this study shows that two KIR populations work collaboratively to govern electrical communication and the spread of vasomotor responses along cerebral arteries.

CaV1.2/CaV3.x channels mediate divergent vasomotor responses in human cerebral arteries.

The regulation of arterial tone is critical in the spatial and temporal control of cerebral blood flow. Voltage-gated Ca(2+) (CaV) channels are key regulators of excitation-contraction coupling in arterial smooth muscle, and thereby of arterial tone. Although L- and T-type CaV channels have been identified in rodent smooth muscle, little is known about the expression and function of specific CaV subtypes in human arteries. Here, we determined which CaV subtypes are present in human cerebral arteries and defined their roles in determining arterial tone. Quantitative polymerase chain reaction and Western blot analysis, respectively, identified mRNA and protein for L- and T-type channels in smooth muscle of cerebral arteries harvested from patients undergoing resection surgery. Analogous to rodents, CaV1.2 (L-type) and CaV3.2 (T-type) α1 subunits were expressed in human cerebral arterial smooth muscle; intriguingly, the CaV3.1 (T-type) subtype present in rodents was replaced with a different T-type isoform, CaV3.3, in humans. Using established pharmacological and electrophysiological tools, we separated and characterized the unique profiles of Ca(2+) channel subtypes. Pressurized vessel myography identified a key role for CaV1.2 and CaV3.3 channels in mediating cerebral arterial constriction, with the former and latter predominating at higher and lower intraluminal pressures, respectively. In contrast, CaV3.2 antagonized arterial tone through downstream regulation of the large-conductance Ca(2+)-activated K(+) channel. Computational analysis indicated that each Ca(2+) channel subtype will uniquely contribute to the dynamic regulation of cerebral blood flow. In conclusion, this study documents the expression of three distinct Ca(2+) channel subtypes in human cerebral arteries and further shows how they act together to orchestrate arterial tone.

Combined effect of sildenafil and guanethidine, propranolol or verapamil on erectile function in rats.

OBJECTIVES: This study aims to further elucidate the role of adrenergic transmission in erection and to highlight whether adrenergic transmission in the penis modulates sildenafil's action. METHODS: Measurement of intracavernosal pressure in the anesthetized rat model. KEY FINDINGS: Guanethidine (3 and 6 mg/kg) potentiated intracavernosal pressure/mean arterial pressure (ICP/MAP) rises in response to cavernous nerve stimulation by 4.375 ± 0.425 and 18.375 ± 1.085% respectively. Propranolol did the opposite. In presence of guanethidine, sildenafil (0.01, 0.1 and 1 mg/kg) potentiated ICP/MAP responses by 81.571 ± 4.918%, 147.83 ± 10.864% and 279.285 ± 23.053% at 1 Hz compared to 22.277 ± 2.139%, 123.571 ± 8.443% and 186.25 ± 13.542% respectively in the absence of guanethidine. Propranolol inhibited the effect sildenafil at all frequencies of stimulation. Verapamil exhibited a pro-erectile action and potentiated the effect of sildenafil (0.01, 0.1 and 1 mg/kg) on erectile responses corresponding to 85.25 ± 6.716%, 146 ± 11.288% and 221.571 ± 19.032% respectively compared to 26.011 ± 1.911%, 87.142 ± 8.73% and 182.2 ± 16.921% in its absence. CONCLUSIONS: This study provides functional evidence that inhibition of sympathetic tone peripherally results in enhancement of erectile function. ß-adrenergic receptors seem to play an important role in erection. The combination of sildenafil and guanethidine or verapamil could have a potential advantage on erectile function but propranolol may mask the effect of sildenafil on erectile function.