Gregory C. Amberg

Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria

1 Pacemaker cells, known as interstitial cells of Cajal (ICC), generate electrical rhythmicity in the gastrointestinal tract. Pacemaker currents in ICC result from the activation of a voltage‐independent, non‐selective cation conductance, but the timing mechanism responsible for periodic activation of the pacemaker current is unknown. 2 Previous studies suggest that pacemaking in ICC is dependent upon metabolic activity 1y1yand1 Ca2+ release from intracellular stores. We tested the hypothesis that mitochondrial Ca2+ handling may underlie the dependence of gastrointestinal pacemaking on oxidative metabolism. 3 Pacemaker currents occurred spontaneously in cultured ICC and were associated with mitochondrial Ca2+ transients. 4 Inhibition of the electrochemical gradient across the inner mitochondrial membrane blocked Ca2+ uptake and pacemaker currents in cultured ICC and blocked slow wave activity in intact gastrointestinal muscles from mouse, dog and guinea‐pig. 5 Pacemaker currents and rhythmic mitochondrial Ca2+ uptake in ICC were also blocked by inhibitors of IP3‐dependent release of Ca2+ from the endoplasmic reticulum and by inhibitors of endoplasmic reticulum Ca2+ reuptake. 6 Our data suggest that integrated Ca2+ handling by endoplasmic reticulum and mitochondria is a prerequisite of electrical pacemaking in the gastrointestinal tract.

Modulation of the molecular composition of large conductance, Ca 2+ activated K + channels in vascular smooth muscle during hypertension

Hypertension is a clinical syndrome characterized by increased vascular tone. However, the molecular mechanisms underlying vascular dysfunction during acquired hypertension remain unresolved. Localized intracellular Ca2+ release events through ryanodine receptors (Ca2+ sparks) in the sarcoplasmic reticulum are tightly coupled to the activation of large-conductance, Ca2+-activated K+ (BK) channels to provide a hyperpolarizing influence that opposes vasoconstriction. In this study we tested the hypothesis that a reduction in Ca2+ spark-BK channel coupling underlies vascular smooth muscle dysfunction during acquired hypertension. We found that in hypertension, expression of the beta1 subunit was decreased relative to the pore-forming alpha subunit of the BK channel. Consequently, the BK channels were functionally uncoupled from Ca2+ sparks. Consistent with this, the contribution of BK channels to vascular tone was reduced during hypertension. We conclude that downregulation of the beta1 subunit of the BK channel contributes to vascular dysfunction in hypertension. These results support the novel concept that changes in BK channel subunit composition regulate arterial smooth muscle function.

Downregulation of the BK Channel β1 Subunit in Genetic Hypertension

Abstract— The molecular mechanisms underlying increased arterial tone during hypertension are unclear. In vascular smooth muscle, localized Ca2+ release events through ryanodine-sensitive channels located in the sarcoplasmic reticulum (Ca2+ sparks) activate large-conductance, Ca2+-sensitive K+ (BK) channels. Ca2+ sparks and BK channels provide a negative feedback mechanism that hyperpolarizes smooth muscle and thereby opposes vasoconstriction. In this study, we examined Ca2+ sparks and BK channel function in Wistar-Kyoto (WKY) rats with borderline hypertension and in spontaneously hypertensive rats (SHR), a widely used genetic model of severe hypertension. We found that the amplitude of spontaneous BK currents in WKY and SHR cells were smaller than in normotensive cells even though Ca2+ sparks were of similar magnitude. BK channels in WKY and SHR cells were less sensitive to physiological changes in intracellular Ca2+ than normotensive cells. Our data indicate that decreased expression of the BK channel &bgr;1 subunit underlies the lower Ca2+ sensitivity of BK channels in SHR and WKY myocytes. We conclude that the lower expression of the &bgr;1 subunit during genetic borderline and severe hypertension reduced BK channel activity by decreasing the sensitivity of these channels to physiological changes in Ca2+. These results support the view that changes in the molecular composition of BK channels may be a fundamental event contributing to the development of vascular dysfunction during hypertension.

AKAP150 Is Required for Stuttering Persistent Ca2+ Sparklets and Angiotensin II–Induced Hypertension

Hypertension is a perplexing multiorgan disease involving renal primary pathology and enhanced angiotensin II vascular reactivity. Here, we report that a novel form of a local Ca2+ signaling in arterial smooth muscle is linked to the development of angiotensin II–induced hypertension. Long openings and reopenings of L-type Ca2+ channels in arterial myocytes produce stuttering persistent Ca2+ sparklets that increase Ca2+ influx and vascular tone. These stuttering persistent Ca2+ sparklets arise from the molecular interactions between the L-type Ca2+ channel and protein kinase C&agr; at only a few subsarcolemmal regions in resistance arteries. We have identified AKAP150 as the key protein, which targets protein kinase C&agr; to the L-type Ca2+ channels and thereby enables its regulatory function. Accordingly, AKAP150 knockout mice (AKAP150−/−) were found to lack persistent Ca2+ sparklets and have lower arterial wall intracellular calcium ([Ca2+]i) and decreased myogenic tone. Furthermore, AKAP150−/− mice were hypotensive and did not develop angiotensin II–induced hypertension. We conclude that local control of L-type Ca2+ channel function is regulated by AKAP150-targeted protein kinase C&agr; signaling, which controls stuttering persistent Ca2+ influx, vascular tone, and blood pressure under physiological conditions and underlies angiotensin II–dependent hypertension.

Mechanisms Underlying Heterogeneous Ca2+ Sparklet Activity in Arterial Smooth Muscle

In arterial smooth muscle, single or small clusters of Ca2+ channels operate in a high probability mode, creating sites of nearly continual Ca2+ influx (called “persistent Ca2+ sparklet” sites). Persistent Ca2+ sparklet activity varies regionally within any given cell. At present, the molecular identity of the Ca2+ channels underlying Ca2+ sparklets and the mechanisms that give rise to their spatial heterogeneity remain unclear. Here, we used total internal reflection fluorescence (TIRF) microscopy to directly investigate these issues. We found that tsA-201 cells expressing L-type Cavα1.2 channels recapitulated the general features of Ca2+ sparklets in cerebral arterial myocytes, including amplitude of quantal event, voltage dependencies, gating modalities, and pharmacology. Furthermore, PKCα activity was required for basal persistent Ca2+ sparklet activity in arterial myocytes and tsA-201 cells. In arterial myocytes, inhibition of protein phosphatase 2A (PP2A) and 2B (PP2B; calcineurin) increased Ca2+ influx by evoking new persistent Ca2+ sparklet sites and by increasing the activity of previously active sites. The actions of PP2A and PP2B inhibition on Ca2+ sparklets required PKC activity, indicating that these phosphatases opposed PKC-mediated phosphorylation. Together, these data unequivocally demonstrate that persistent Ca2+ sparklet activity is a fundamental property of L-type Ca2+ channels when associated with PKC. Our findings support a novel model in which the gating modality of L-type Ca2+ channels vary regionally within a cell depending on the relative activities of nearby PKCα, PP2A, and PP2B.

Activation of NFATc3 Down-regulates the β1 Subunit of Large Conductance, Calcium-activated K+ Channels in Arterial Smooth Muscle and Contributes to Hypertension

Large conductance, Ca2+-activated K+ (BK) channels modulate the excitability and contractile state of arterial smooth muscle. Recently, we demonstrated that during hypertension, expression of the accessory β1 subunit was decreased relative to the pore-forming α subunit of the BK channel. Reduced β1 subunit expression resulted in BK channels with impaired function due to lowered sensitivity to Ca2+. Here, we tested the hypothesis that activation of the calcineurin/NFATc3 signaling pathway down-regulates β1 expression during angiotensin II-induced hypertension. Consistent with this hypothesis, we found that in vivo administration of angiotensin II-activated calcineurin/NFATc3 signaling in arterial smooth muscle. During angiotensin II infusion, arterial smooth muscle BK channel function was decreased in wild type (WT) but not in NFATc3 null (NFATc3-/-) mice. Accordingly, β1 expression was decreased in WT but not in NFATc3-/- arteries. Angiotensin II-induced down-regulation of the β1 subunit required Ca2+ influx via L-type Ca2+ channels. However, in the absence of angiotensin II, moderate elevation of [Ca2+]i alone was not sufficient to activate NFAT transcriptional activity and, thus, decrease β1 subunit expression. Importantly, angiotensin II infusion increased systemic blood pressure to a lower extent in NFATc3-/- than in WT mice, indicating that this transcription factor is required for the development of severe hypertension during chronic angiotensin II signaling activation. We conclude that activation of calcineurin and NFATc3 during sustained angiotensin II signaling down-regulates the expression of the β1 subunit of the BK channel, which in turn contributes to arterial dysfunction and the development of hypertension.

The control of Ca2+ influx and NFATc3 signaling in arterial smooth muscle during hypertension

Many excitable cells express L-type Ca2+ channels (LTCCs), which participate in physiological and pathophysiological processes ranging from memory, secretion, and contraction to epilepsy, heart failure, and hypertension. Clusters of LTCCs can operate in a PKCα-dependent, high open probability mode that generates sites of sustained Ca2+ influx called “persistent Ca2+ sparklets.” Although increased LTCC activity is necessary for the development of vascular dysfunction during hypertension, the mechanisms leading to increased LTCC function are unclear. Here, we tested the hypothesis that increased PKCα and persistent Ca2+ sparklet activity contributes to arterial dysfunction during hypertension. We found that PKCα and persistent Ca2+ sparklet activity is indeed increased in arterial myocytes during hypertension. Furthermore, in human arterial myocytes, PKCα-dependent persistent Ca2+ sparklets activated the prohypertensive calcineurin/NFATc3 signaling cascade. These events culminated in three hallmark signs of hypertension-associated vascular dysfunction: increased Ca2+ entry, elevated arterial [Ca2+]i, and enhanced myogenic tone. Consistent with these observations, we show that PKCα ablation is protective against the development of angiotensin II-induced hypertension. These data support a model in which persistent Ca2+ sparklets, PKCα, and calcineurin form a subcellular signaling triad controlling NFATc3-dependent gene expression, arterial function, and blood pressure. Because of the ubiquity of these proteins, this model may represent a general signaling pathway controlling gene expression and cellular function.

Calcium sparklets regulate local and global calcium in murine arterial smooth muscle

In arterial smooth muscle, protein kinase Cα (PKCα) coerces discrete clusters of L‐type Ca2+ channels to operate in a high open probability mode, resulting in subcellular domains of nearly continual Ca2+ influx called ‘persistent Ca2+ sparklets’. Our previous work suggested that steady‐state Ca2+ entry into arterial myocytes, and thus global [Ca2+]i, is regulated by Ca2+ influx through clusters of L‐type Ca2+ channels operating in this persistently active mode in addition to openings of solitary channels functioning in a low‐activity mode. Here, we provide the first direct evidence supporting this ‘Ca2+ sparklet’ model of Ca2+ influx at a physiological membrane potential and external Ca2+ concentration. In support of this model, we found that persistent Ca2+ sparklets produced local and global elevations in [Ca2+]i. Membrane depolarization increased Ca2+ influx via low‐activity and high‐activity persistent Ca2+ sparklets. Our data indicate that Ca2+ entering arterial smooth muscle through persistent Ca2+ sparklets accounts for approximately 50% of the total dihydropyridine‐sensitive (i.e. L‐type Ca2+ channel) Ca2+ influx at a physiologically relevant membrane potential (−40 mV) and external Ca2+ concentration (2 mm). Consistent with this, inhibition of basal PKCα‐dependent persistent Ca2+ sparklets decreased [Ca2+]i by about 50% in isolated arterial myocytes and intact pressurized arteries. Taken together, these data support the conclusion that in arterial smooth muscle steady‐state Ca2+ entry and global [Ca2+]i are regulated by low‐activity and PKCα‐dependent high‐activity persistent Ca2+ sparklets.

Pharmacological inhibition of TRPM4 hyperpolarizes vascular smooth muscle

The contractile state of vascular smooth muscle cells is regulated by small changes in membrane potential that gate voltage-dependent calcium channels. The melastatin transient receptor potential (TRP) channel TRPM4 is a critical mediator of pressure-induced membrane depolarization and arterial constriction. A recent study shows that the tricyclic compound 9-phenanthrol inhibits TRPM4, but not the related channel TRPM5. The current study investigated the specificity of 9-phenanthrol and the effects of the compound on pressure-induced smooth muscle depolarization and arterial constriction. Patch-clamp electrophysiology revealed that 9-phenanthrol blocks native TRPM4 currents in freshly isolated smooth muscle cells in a concentration-dependent manner (IC(50) = 10.6 μM). 9-Phenanthrol (30 μM) had no effect on maximum evoked currents in human embryonic kidney cells expressing recombinant TRPC3 or TRPC6 channels. Large-conductance Ca(2+)-activated K(+), voltage-dependent K(+), inwardly rectifying K(+), and voltage-dependent Ca(2+) channel activity in native cerebral artery myocytes was not altered by administration of 9-phenanthrol (30 μM). Using intracellular microelectrodes to record smooth muscle membrane potential in isolated cerebral arteries pressurized to 70 mmHg, we found that 9-phenanthrol (30 μM) reversibly hyperpolarized the membrane from ∼-40 mV to ∼-70 mV. In addition, we found that myogenic tone was reversibly abolished when vessels were exposed to 9-phenanthrol. These data demonstrate that 9-phenanthrol is useful for studying the functional significance of TRPM4 in vascular smooth muscle cells and that TRPM4 is an important regulator of smooth muscle cell membrane depolarization and arterial constriction in response to intraluminal pressure.

Ca2+ release from the sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells

The melastatin transient receptor potential (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. Activation of the channel is Ca(2+)-dependent, but prolonged exposure to high (>1 microM) levels of intracellular Ca(2+) causes rapid (within approximately 2 min) desensitization of TRPM4 currents under conventional whole cell and inside-out patch-clamp conditions. The goal of the present study was to establish a novel method to record sustained TRPM4 currents in smooth muscle cells under near-physiological conditions. Using the amphotericin B-perforated patch-clamp technique, we recorded and characterized sustained (up to 30 min) transient inward cation currents (TICCs) in freshly isolated cerebral artery myocytes. In symmetrical cation solutions, TICCs reversed at 0 mV and had an apparent unitary conductance of 25 pS. Replacement of extracellular Na(+) with the nonpermeable cation N-methyl-d-glucamine abolished the current. TICC activity was attenuated by the TRPM4 blockers fluflenamic acid and 9-phenanthrol. Selective silencing of TRPM4 expression using small interfering RNA diminished TICC activity, suggesting that the molecular identity of the responsible ion channel is TRPM4. We used the perforated patch-clamp method to test the hypothesis that TRPM4 is activated by intracellular Ca(2+) signaling events. We found that TICC activity is independent of Ca(2+) influx and ryanodine receptor activity but is attenuated by sarco(endo)plasmic reticulum Ca(2+)-ATPase inhibition and blockade of inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release from the sarcoplasmic reticulum. Our findings suggest that TRPM4 channels in cerebral artery myocytes are regulated by Ca(2+) release from inositol 1,4,5-trisphosphate receptor on the sarcoplasmic reticulum.