Osama F. Harraz

Identification of L- and T-type Ca2+ channels in rat cerebral arteries: role in myogenic tone development

L-type Ca(2+) channels are broadly expressed in arterial smooth muscle cells, and their voltage-dependent properties are important in tone development. Recent studies have noted that these Ca(2+) channels are not singularly expressed in vascular tissue and that other subtypes are likely present. In this study, we ascertained which voltage-gated Ca(2+) channels are expressed in rat cerebral arterial smooth muscle and determined their contribution to the myogenic response. mRNA analysis revealed that the α(1)-subunit of L-type (Ca(v)1.2) and T-type (Ca(v)3.1 and Ca(v)3.2) Ca(2+) channels are present in isolated smooth muscle cells. Western blot analysis subsequently confirmed protein expression in whole arteries. With the use of patch clamp electrophysiology, nifedipine-sensitive and -insensitive Ba(2+) currents were isolated and each were shown to retain electrical characteristics consistent with L- and T-type Ca(2+) channels. The nifedipine-insensitive Ba(2+) current was blocked by mibefradil, kurtoxin, and efonidpine, T-type Ca(2+) channel inhibitors. Pressure myography revealed that L-type Ca(2+) channel inhibition reduced tone at 20 and 80 mmHg, with the greatest effect at high pressure when the vessel is depolarized. In comparison, the effect of T-type Ca(2+) channel blockade on myogenic tone was more limited, with their greatest effect at low pressure where vessels are hyperpolarized. Blood flow modeling revealed that the vasomotor responses induced by T-type Ca(2+) blockade could alter arterial flow by ∼20-50%. Overall, our findings indicate that L- and T-type Ca(2+) channels are expressed in cerebral arterial smooth muscle and can be electrically isolated from one another. Both conductances contribute to myogenic tone, although their overall contribution is unequal.

Nitric Oxide Suppresses Vascular Voltage-Gated T-Type Ca2+ Channels Through cGMP/PKG Signaling

Recent reports have noted that T-type Ca2+ channels (CaV3.x) are expressed in vascular smooth muscle and are potential targets of regulation. In this study, we examined whether and by what mechanism nitric oxide (NO), a key vasodilator, influences this conductance. Using patch-clamp electrophysiology and rat cerebral arterial smooth muscle cells, we monitored an inward Ba2+ current that was divisible into a nifedipine-sensitive and -insensitive component. The latter was abolished by T-type channel blocker and displayed classic T-type properties including faster activation and steady-state inactivation at hyperpolarized potentials. NO donors (sodium nitroprusside, S-nitroso-N-acetyl-dl-penicillamine), along with activators of protein kinase G (PKG) signaling, suppressed T-type currents. Inhibitors of guanylyl cyclase/PKG {1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and KT5823, respectively}, had no effect on basal currents; KT5823 did, however, mask T-type Ca2+ channel current inhibition by NO/PKG. Functional experiments confirmed an inhibitory effect for NO on the T-type contribution to cerebral arterial myogenic tone. Cumulatively, our findings support the view that T-type Ca2+ channels are a regulatory target of vasodilatory signaling pathways. This targeting will influence Ca2+ dynamics and consequent tone development in the cerebral circulation.

CaV3.2 Channels and the Induction of Negative Feedback in Cerebral Arteries

Rationale: T-type (CaV3.1/CaV3.2) Ca2+ channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. Objective: This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca2+ sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca2+-activated K+ channels. Methods and Results: Micromolar Ni2+, an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2−/− arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca2+ influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca2+-induced Ca2+ release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca2+ imaging and perforated patch clamp electrophysiology demonstrated that Ni2+ suppressed Ca2+ sparks and consequently spontaneous transient outward K+ currents, large-conductance Ca2+-activated K+ channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca2+-activated K+ channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni2+. Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. Conclusions: These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor–mediated Ca2+ sparks, enabling large-conductance Ca2+-activated K+ channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

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

Human cerebral arteries contain three Ca2+ channel subtypes with distinct physiological roles.

STIM1-mediated bidirectional regulation of Ca2+ entry through voltage-gated calcium channels (VGCC) and calcium-release activated channels (CRAC)

The spatial and temporal regulation of cellular calcium signals is modulated via two main Ca2+ entry routes. Voltage-gated Ca2+ channels (VGCC) and Ca2+-release activated channels (CRAC) enable Ca2+ flow into electrically excitable and non-excitable cells, respectively. VGCC are well characterized transducers of electrical activity that allow Ca2+ signaling into the cell in response to action potentials or subthreshold depolarizing stimuli. The identification of STromal Interaction Molecule (STIM) and Orai proteins has provided significant insights into the understanding of CRAC function and regulation. This review will summarize the current state of knowledge of STIM-Orai interaction and their contribution to cellular Ca2+ handling mechanisms. We will then discuss the bidirectional actions of STIM1 on VGCC and CRAC. In contrast to the stimulatory role of STIM1 on Orai channel activity that facilitates Ca2+ entry, recent reports indicated the ability of STIM1 to suppress VGCC activity. This new concept changes our traditional understanding of Ca2+ handling mechanisms and highlights the existence of dynamically regulated signaling complexes of surface expressed ion channels and intracellular store membrane-embedded Ca2+ sensors. Overall, STIM1 is emerging as a new class of regulatory proteins that fine-tunes Ca2+ entry in response to endoplasmic/sarcoplasmic reticulum stress.

Protein kinase A regulation of T-type Ca2+ channels in rat cerebral arterial smooth muscle

Summary Recent investigations have identified that T-type Ca2+ channels (CaV3.x) are expressed in rat cerebral arterial smooth muscle. In the study reported here, we isolated the T-type conductance, differentiated the current into the CaV3.1/CaV3.2 subtypes and determined whether they are subject to protein kinase regulation. Using patch clamp electrophysiology, whole-cell Ba2+ current was monitored and initially subdivided into nifedipine-sensitive and -insensitive components. The latter conductance was abolished by T-type Ca2+ channel blockers and was faster with leftward shifted activation/inactivation properties, reminiscent of a T-type channel. Approximately 60% of this T-type conductance was blocked by 50 µM Ni2+, a concentration that selectively interferes with CaV3.2 channels. Subsequent work revealed that the whole-cell T-type conductance was subject to protein kinase A (PKA) modulation. Specifically, positive PKA modulators (db-cAMP, forskolin, isoproterenol) suppressed T-type currents and evoked a hyperpolarized shift in steady-state inactivation. Blocking PKA (with KT5720) masked this suppression without altering the basal T-type conductance. A similar effect was observed with stHt31, a peptide inhibitor of A-kinase anchoring proteins. A final set of experiments revealed that PKA-induced suppression targeted the CaV3.2 subtype. In summary, this study revealed that a T-type Ca2+ channel conductance can be isolated in arterial smooth muscle, and differentiated into CaV3.1 and CaV3.2 components. It also showed that vasodilatory signaling cascades inhibit this conductance by targeting CaV3.2. Such targeting would impact Ca2+ dynamics and consequent tone regulation in the cerebral circulation.

Genetic Ablation of CaV3.2 Channels Enhances the Arterial Myogenic Response by Modulating the RyR-BKCa Axis

Objective—In resistance arteries, there is an emerging view that smooth muscle CaV3.2 channels restrain arterial constriction through a feedback response involving the large-conductance Ca2+-activated K+ channel (BKCa). Here, we used wild-type and CaV3.2 knockout (CaV3.2−/−) mice to definitively test whether CaV3.2 moderates myogenic tone in mesenteric arteries via the CaV3.2-ryanodine receptor-BKCa axis and whether this regulatory mechanism influences blood pressure regulation. Approach and Results—Using pressurized vessel myography, CaV3.2−/− 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 CaV3.2 blocker, to either constrict arteries or suppress T-type currents in CaV3.2−/− smooth muscle cells. The frequency of BKCa-induced spontaneous transient outward K+ currents dropped in wild-type but not in knockout arterial smooth muscle cells upon the pharmacological suppression of CaV3.2 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 CaV3.2 channel moderated myogenic constriction of resistance arteries, the blood pressure measurements of CaV3.2−/− and wild-type animals were similar. Conclusions—Overall, our findings establish a negative feedback mechanism of the myogenic response in which CaV3.2 channel modulates downstream ryanodine receptor-BKCa to hyperpolarize and relax arteries.

T‐Type Ca2+ Channels in Cerebral Arteries: Approaches, Hypotheses, and Speculation

Cerebral blood flow is controlled by a network of resistance arteries that dilate and constrict to mechanical and chemical stimuli. Vasoactive stimuli influence arterial diameter through alterations in resting membrane potential and the influx of Ca2+ through voltage‐gated Ca2+ channels. Historically, L‐type Ca2+ channels were thought to be solely expressed in cerebral arterial smooth muscle. Recent studies have, however, challenged this perspective, by providing evidence of T‐type Ca2+ channels in vascular tissues. This perspective piece will introduce T‐type Ca2+ channels, their electrophysiological properties, and potential roles in arterial tone development. We begin with a brief overview of Ca2+ channels and a discussion of the approaches used to isolate this elusive conductance. We will then speculate on how the two T‐type Ca2+ channels expressed in cerebral arterial smooth muscle might differentially influence arterial tone. This discovery of T‐type Ca2+ channels alters our traditional understanding of Ca2+ dynamics in vascular tissue and fosters new avenues of research and insight into the basis of arterial tone development.