Ca(V)3.2 channels and the induction of negative feedback in cerebral arteries.

RATIONALE: T-type (CaV3.1/CaV3.2) Ca(2+) 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 Ca(2+) sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca(2+)-activated K(+) channels. METHODS AND RESULTS: Micromolar Ni(2+), 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 Ca(2+) influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca(2+)-induced Ca(2+) release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca(2+) imaging and perforated patch clamp electrophysiology demonstrated that Ni(2+) suppressed Ca(2+) sparks and consequently spontaneous transient outward K(+) currents, large-conductance Ca(2+)-activated K(+) channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca(2+)-activated K(+) channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni(2+). 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 Ca(2+) sparks, enabling large-conductance Ca(2+)-activated K(+) channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

Implications of αvß3 Integrin Signaling in the Regulation of Ca2+ Waves and Myogenic Tone in Cerebral Arteries.

OBJECTIVE: The myogenic response is central to blood flow regulation in the brain. Its induction is tied to elevated cytosolic [Ca(2+)], a response primarily driven by voltage-gated Ca(2+) channels and secondarily by Ca(2+) wave production. Although the signaling events leading to the former are well studied, those driving Ca(2+) waves remain uncertain. APPROACH AND RESULTS: We postulated that αvß3 integrin signaling is integral to the generation of pressure-induced Ca(2+) waves and cerebral arterial tone. This hypothesis was tested in rat cerebral arteries using the synergistic strengths of pressure myography, rapid Ca(2+) imaging, and Western blot analysis. GRGDSP, a peptide that preferentially blocks αvß3 integrin, attenuated myogenic tone, indicating the modest role for sarcoplasmic reticulum Ca(2+) release in myogenic tone generation. The RGD peptide was subsequently shown to impair Ca(2+) wave generation and myosin light chain 20 (MLC20) phosphorylation, the latter of which was attributed to the modulation of MLC kinase and MLC phosphatase via MYPT1-T855 phosphorylation. Subsequent experiments revealed that elevated pressure enhanced phospholipase Cγ1 phosphorylation in an RGD-dependent manner and that phospholipase C inhibition attenuated Ca(2+) wave generation. Direct inhibition of inositol 1, 4, 5-triphosphate receptors also impaired Ca(2+) wave generation, myogenic tone, and MLC20 phosphorylation, partly through the T-855 phosphorylation site of MYPT1. CONCLUSIONS: Our investigation reveals a hitherto unknown role for αvß3 integrin as a cerebral arterial pressure sensor. The membrane receptor facilitates Ca(2+) wave generation through a signaling cascade, involving phospholipase Cγ1, inositol 1,3,4 triphosphate production, and inositol 1, 4, 5-triphosphate receptor activation. These discrete asynchronous Ca(2+) events facilitate MLC20 phosphorylation and, in part, myogenic tone by influencing both MLC kinase and MLC phosphatase activity.

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.

Intravascular pressure augments cerebral arterial constriction by inducing voltage-insensitive Ca2+ waves.

This study examined whether elevated intravascular pressure stimulates asynchronous Ca(2+) waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic tone development. The endothelium was removed from rat cerebral arteries, which were then mounted in an arteriograph, pressurized (20-100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (V(M)) were monitored using conventional techniques; Ca(2+) wave generation and myosin light chain (MLC(20))/MYPT1 (myosin phosphatase targeting subunit) phosphorylation were assessed by confocal microscopy and Western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing asynchronous Ca(2+) waves as well as event frequency. Ca(2+) wave augmentation occurred primarily at lower intravascular pressures (<60 mmHg) and ryanodine, a plant alkaloid that depletes the sarcoplasmic reticulum (SR) of Ca(2+), eliminated these events. Ca(2+) wave generation was voltage insensitive as Ca(2+) channel blockade and perturbations in extracellular [K(+)] had little effect on measured parameters. Ryanodine-induced inhibition of Ca(2+) waves attenuated myogenic tone and MLC(20) phosphorylation without altering arterial V(M). Thapsigargin, an SR Ca(2+)-ATPase inhibitor also attenuated Ca(2+) waves, pressure-induced constriction and MLC(20) phosphorylation. The SR-driven component of the myogenic response was proportionally greater at lower intravascular pressures and subsequent MYPT1 phosphorylation measures revealed that SR Ca(2+) waves facilitated pressure-induced MLC(20) phosphorylation through mechanisms that include myosin light chain phosphatase inhibition. Cumulatively, our findings show that mechanical stimuli augment Ca(2+) wave generation in arterial smooth muscle and that these transient events facilitate tone development particularly at lower intravascular pressures by providing a proportion of the Ca(2+) required to directly control MLC(20) phosphorylation.

Thrombin enhances NGF-mediated neurite extension via increased and sustained activation of p44/42 MAPK and p38 MAPK.

Rapid neurite remodeling is fundamental to nervous system development and plasticity. It involves neurite extension that is regulated by NGF through PI3K/AKT, p44/42 MAPK and p38 MAPK. It also involves neurite retraction that is regulated by the serine protease, thrombin. However, the intracellular signaling pathway by which thrombin causes neurite retraction is unknown. Using the PC12 neuronal cell model, we demonstrate that thrombin utilizes the PI3K/AKT pathway for neurite retraction in NGF-differentiated cells. Interestingly, however, we found that thrombin enhances NGF-induced neurite extension in differentiating cells. This is achieved through increased and sustained activation of p44/42 MAPK and p38 MAPK. Thus, thrombin elicits opposing effects in differentiated and differentiating cells through activation of distinct signaling pathways: neurite retraction in differentiated cells via PI3K/AKT, and neurite extension in differentiating cells via p44/42 MAPK and p38 MAPK. These findings, which also point to a novel cooperative role between thrombin and NGF, have significant implications in the development of the nervous system and the disease processes that afflicts it as well as in the potential of combined thrombin and NGF therapy for impaired learning and memory, and spinal cord injury which all require neurite extension and remodeling.