Group III metabotropic glutamate receptors regulate hypothalamic presympathetic neurons through opposing presynaptic and postsynaptic actions in hypertension.

The hypothalamic paraventricular nucleus (PVN) plays a major role in generating increased sympathetic output in hypertension. Although group III metabotropic glutamate receptors (mGluRs) are expressed in the hypothalamus, little is known about their contribution to regulating PVN presympathetic neurons in hypertension. Here we show that activating group III mGluRs with L-2-amino-4-phosphonobutyric acid (L-AP4) consistently inhibited the firing activity of spinally projecting PVN neurons in normotensive rats. However, in spontaneously hypertensive rats (SHRs), L-AP4 inhibited 45% of PVN neurons but excited 37%. L-AP4 significantly reduced glutamatergic and GABAergic input to PVN neurons in both groups. Blocking postsynaptic G protein signaling eliminated the excitatory but not the inhibitory effect of L-AP4 on PVN neurons in SHRs. Remarkably, prior activation of group I mGluRs converted the L-AP4 effect from inhibitory to excitatory in PVN neurons, and L-AP4 consistently inhibited PVN neurons when mGluR5 was blocked in SHRs. Furthermore, the expression level of mGluR4 and mGluR6 in the PVN was significantly higher in SHRs than in normotensive rats. Microinjection of L-AP4 into the PVN decreased blood pressure and lumbar sympathetic nerve discharges in normotensive rats and SHRs. Additionally, blocking group I mGluRs in the PVN potentiated L-AP4's sympathoinhibitory effect in SHRs. Therefore, activation of presynaptic group III mGluRs inhibits the excitability of PVN presympathetic neurons to attenuate sympathetic vasomotor activity. Through crosstalk with mGluR5, postsynaptic group III mGluR stimulation paradoxically excites PVN presympathetic neurons in SHRs. Concurrently blocking mGluR5 and activating group III mGluRs in the PVN can effectively reduce sympathetic outflow in hypertension.

Protein kinase CK2 contributes to diminished small conductance Ca2+-activated K+ channel activity of hypothalamic pre-sympathetic neurons in hypertension.

Small conductance calcium-activated K(+) (SK) channels regulate neuronal excitability. However, little is known about changes in SK channel activity of pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) in essential hypertension. SK channels, calmodulin, and casein kinase II (CK2) form a molecular complex. Because CK2 is up-regulated in the PVN in spontaneously hypertensive rats (SHRs), we hypothesized that CK2 increases calmodulin phosphorylation and contributes to diminished SK channel activity in PVN pre-sympathetic neurons in SHRs. Perforated whole-cell recordings were performed on retrogradely labeled spinally projecting PVN neurons in Wistar-Kyoto (WKY) rats and SHRs. Blocking SK channels with apamin significantly increased the firing rate of PVN neurons in WKY rats but not in SHRs. CK2 inhibition restored the stimulatory effect of apamin on the firing activity of PVN neurons in SHRs. Furthermore, apamin-sensitive SK currents and depolarization-induced medium after-hyperpolarization potentials of PVN neurons were significantly larger in WKY rats than in SHRs. CK2 inhibition significantly increased the SK channel current and medium after-depolarization potential of PVN neurons in SHRs. In addition, CK2-mediated calmodulin phosphorylation level in the PVN was significantly higher in SHRs than in WKY rats. Although SK3 was detected in the PVN, its expression level did not differ significantly between SHRs and WKY rats. Our findings suggest that CK2-mediated calmodulin phosphorylation is increased and contributes to diminished SK channel function of PVN pre-sympathetic neurons in SHRs. This information advances our understanding of the mechanisms underlying hyperactivity of PVN pre-sympathetic neurons and increased sympathetic vasomotor tone in hypertension. Small conductance calcium-activated K(+) (SK) channels, calmodulin, and protein kinase CK2 form a molecular complex and regulate neuronal excitability. Our study suggests that augmented CK2 activity in hypertension can increase calmodulin (CaM) phosphorylation, which leads to diminished SK channel function in pre-sympathetic neurons. Diminished SK channel activity plays a role in hyperactivity of pre-sympathetic neurons in the hypothalamus in hypertension.

mGluR5 Upregulation increases excitability of hypothalamic presympathetic neurons through NMDA receptor trafficking in spontaneously hypertensive rats.

The hypothalamic paraventricular nucleus (PVN) is critically involved in elevated sympathetic output and the development of hypertension. However, changes in group I metabotropic glutamate receptors (mGluR1 and mGluR5) and their relevance to the hyperactivity of PVN presympathetic neurons in hypertension remain unclear. Here, we found that selectively blocking mGluR5 significantly reduced the basal firing activity of spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs), but not in normotensive Wistar-Kyoto (WKY) rats. However, blocking mGluR1 had no effect on the firing activity of PVN neurons in either group. The mRNA and protein levels of mGluR5 in the PVN and rostral ventrolateral medulla were significantly higher in SHRs than in WKY rats. The group I mGluR selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) similarly increased the firing activity of PVN neurons in WKY rats and SHRs. In addition, blocking NMDA receptors (NMDARs) through bath application or intracellular dialysis not only decreased the basal firing in SHRs, but also eliminated DHPG-induced excitation of spinally projecting PVN neurons. DHPG significantly increased the amplitude of NMDAR currents without changing their decay kinetics. Interestingly, DHPG still increased the amplitude of NMDAR currents and caused reappearance of functional NMDAR channels after initially blocking NMDARs. In addition, protein kinase C (PKC) inhibition or intracellular dialysis with synaptosomal-associated protein of 25 kDa (SNAP-25)-blocking peptide abolished DHPG-induced increases in NMDAR currents of PVN neurons in SHRs. Our findings suggest that mGluR5 in the PVN is upregulated in hypertension and contributes to the hyperactivity of PVN presympathetic neurons through PKC- and SNAP-25-mediated surface expression of NMDARs.

The voltage-dependent L-type Ca2+ (CaV1.2) channel C-terminus fragment is a bi-modal vasodilator.

Voltage-dependent L-type Ca(2+) channels (CaV1.2) are the primary Ca(2+) entry pathway in vascular smooth muscle cells (myocytes). CaV1.2 channels control systemic blood pressure and organ blood flow and are pathologically altered in vascular diseases, which modifies vessel contractility. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt). Previous studies in cardiac myocytes and neurons have identified CCt as both a transcription factor and CaV1.2 channel inhibitor, with different signalling mechanisms proposed to underlie some of these effects. CCt existence and physiological functions in arterial myocytes are unclear, but important to study given the functional significance of CaV1.2 channels. Here, we show that CCt exists in myocytes of both rat and human resistance-size cerebral arteries, where it locates to both the nucleus and plasma membrane. Recombinant CCt expression in arterial myocytes inhibited CaV1.2 transcription and reduced CaV1.2 protein. CCt induced a depolarizing shift in the voltage dependence of both CaV1.2 current activation and inactivation, and reduced non-inactivating current in myocytes. Recombinant truncated CCt lacking a putative nuclear localization sequence (92CCt) did not locate to the nucleus and had no effect on arterial CaV1.2 transcription or protein. However, 92CCt shifted the voltage dependence of CaV1.2 activation and inactivation similarly to CCt. CCt and 92CCt both inhibited pressure- and depolarization-induced vasoconstriction, although CCt was a far more effective vasodilator. These data demonstrate that endogenous CCt exists and reduces both CaV1.2 channel expression and voltage sensitivity in arterial myocytes. Thus, CCt is a bi-modal vasodilator.

Transcriptional upregulation of α2δ-1 elevates arterial smooth muscle cell voltage-dependent Ca2+ channel surface expression and cerebrovascular constriction in genetic hypertension.

A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca2+ (CaV1.2) currents that induces pathological vasoconstriction. CaV1.2 channels are heteromeric complexes composed of a pore-forming CaV1.2α1 with auxiliary α2δ and ß subunits. Molecular mechanisms that elevate CaV1.2 currents during hypertension and the potential contribution of CaV1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α2δ subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats was associated with an unequal elevation in α2δ-1 and CaV1.2α1 mRNA and protein in cerebral artery myocytes, with α2δ-1 increasing more than CaV1.2α1. Other α2δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive spontaneously hypertensive rats displayed higher surface-localized α2δ-1 and CaV1.2α1 proteins, surface α2δ-1:CaV1.2α1 ratio, CaV1.2 current density and noninactivating current, and pressure- and depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α2δ-1 ligand, did not alter α2δ-1 or CaV1.2α1 total protein but normalized α2δ-1 and CaV1.2α1 surface expression, surface α2δ-1:CaV1.2α1, CaV1.2 current density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in α2δ-1 expression that promotes surface trafficking of CaV1.2 channels in cerebral artery myocytes. This leads to an increase in CaV1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α2δ-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension.

Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels.

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels.

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells.

Plasma membrane large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK(Ca) channel regulation by IP(3) and IP(3)Rs in rat and mouse cerebral artery smooth muscle cells. IP(3) activated BK(Ca) channels both in intact cells and in excised inside-out membrane patches. IP(3) caused concentration-dependent BK(Ca) channel activation with an apparent dissociation constant (K(d)) of approximately 4 microM at physiological voltage (-40 mV) and intracellular Ca(2+) concentration ([Ca(2+)](i); 10 microM). IP(3) also caused a leftward-shift in BK(Ca) channel apparent Ca(2+) sensitivity and reduced the K(d) for free [Ca(2+)](i) from approximately 20 to 12 microM, but did not alter the slope or maximal P(o). BAPTA, a fast Ca(2+) buffer, or an elevation in extracellular Ca(2+) concentration did not alter IP(3)-induced BK(Ca) channel activation. Heparin, an IP(3)R inhibitor, and a monoclonal type 1 IP(3)R (IP(3)R1) antibody blocked IP(3)-induced BK(Ca) channel activation. Adenophostin A, an IP(3)R agonist, also activated BK(Ca) channels. IP(3) activated BK(Ca) channels in inside-out patches from wild-type (IP(3)R1(+/+)) mouse arterial smooth muscle cells, but had no effect on BK(Ca) channels of IP(3)R1-deficient (IP(3)R1(-/-)) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP(3)R1 is located in close spatial proximity to BK(Ca) alpha subunits. The IP(3)R1 monoclonal antibody coimmunoprecipitated IP(3)R1 and BK(Ca) channel alpha and beta1 subunits from cerebral arteries. In summary, data indicate that IP(3)R1 activation elevates BK(Ca) channel apparent Ca(2+) sensitivity through local molecular coupling in arterial smooth muscle cells.

Alternative splicing of Cav1.2 channel exons in smooth muscle cells of resistance-size arteries generates currents with unique electrophysiological properties.

Voltage-dependent calcium (Ca(2+), Ca(V)1.2) channels are the primary Ca(2+) entry pathway in smooth muscle cells of resistance-size (myogenic) arteries, but their molecular identity remains unclear. Here we identified and quantified Ca(V)1.2 alpha(1)-subunit splice variation in myocytes of rat resistance-size (100-200 microm diameter) cerebral arteries. Full-length clones containing either exon 1b or the recently identified exon 1c exhibited additional primary splice variation at exons 9*, 21/22, 31/32, and +/- 33. Real-time PCR confirmed the findings from full-length clones and indicated that the major Ca(V)1.2 variant contained exons 1c, 8, 21, and 32+33, with approximately 57% containing 9*. Exon 9* was more prevalent in clones containing 1c (72%) than in those containing 1b (33%), suggesting exon-selective combinatorial splicing. To examine the functional significance of this splicing profile, membrane currents produced by each of the four exon 1b/c/ +/- 9* variants were characterized following transfection in HEK293 cells. Exon 1c and 9* caused similar hyperpolarizing shifts in both current-voltage relationships and voltage-dependent activation of currents. Furthermore, exon 9* induced a hyperpolarizing shift only in the voltage-dependent activation of channels containing exon 1b, but not in those containing exon 1c. In contrast, exon 1b, 1c, or +9* did not alter voltage-dependent inactivation. In summary, we have identified the Ca(V)1.2 alpha(1)-subunit splice variant population that is expressed in myocytes of resistance-size arteries and the unique electrophysiological properties of recombinant channels formed by exon 1 and 9* variation. The predominance of exon 1c and 9* in smooth muscle cell Ca(V)1.2 channels causes a hyperpolarizing shift in the voltage sensitivity of currents toward the physiological arterial voltage range.

CNTF-evoked activation of JAK and ERK mediates the functional expression of T-type Ca2+ channels in chicken nodose neurons.

Culture of chicken nodose neurons with CNTF but not BDNF causes a significant increase in T-type Ca(2+) channel expression. CNTF-induced channel expression requires 12 h stimulation to reach maximal expression and is not affected by inhibition of protein synthesis, suggesting the involvement of a post-translational mechanism. In this study, we have investigated the biochemical mechanism responsible for the CNTF-dependent stimulation of T-type channel expression in nodose neurons. Stimulation of nodose neurons with CNTF evoked a considerable increase in signal transducer and activator of transcription (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation. CNTF-evoked ERK phosphorylation was transient whereas BDNF-evoked activation of ERK was sustained. Pre-treatment of nodose neurons with the Janus tyrosine kinase (JAK) inhibitor P6 blocked STAT3 and ERK phosphorylation, whereas the ERK inhibitor U0126 prevented ERK activation but not STAT3 phosphorylation. Both P6 and U0126 inhibited the stimulatory effect of CNTF on T-type channel expression. Inhibition of STAT3 activation by the selective blocker stattic has no effect on ERK phosphorylation and T-type channel expression. These results indicate that CNTF-evoked stimulation of T-type Ca(2+) channel expression in chicken nodose neurons requires JAK-dependent ERK signaling. A cardiac tissue extract derived from E20 chicken heart was also effective in promoting T-type Ca(2+) channel expression and STAT3 and ERK phosphorylation. The ability of the heart extract to stimulate JAK/STAT and ERK activation was developmentally regulated. These findings provide further support to the idea that CNTF or a CNTF-like factor mediates normal expression of T-type channels.

Expression pattern of T-type Ca(2+) channels in embryonic chick nodose ganglion neurons.

In this study we have characterized the functional expression of T-type Ca(2+) channels in developing chick nodose neurons, a population of placode-derived sensory neurons innervating the heart and various visceral organs. Voltage-gated Ca(2+) currents were measured using whole cell patch clamp recordings in neurons acutely isolated between embryonic day (E) 7 and E20, prior to hatching. E7 nodose neurons express relatively large high voltage-activated (HVA) Ca(2+) currents. HVA current density progressively increases between E7 and E17. T-type Ca(2+) currents were restricted to a few nodose neurons between E7 and E10 but were present in approximately 60% of nodose neurons by E17. T-type Ca(2+) channels regulate the response of nodose neurons to injection of hyperpolarizing currents, but do not have any effect on the action potential waveform. Nickel ions blocked T-type Ca(2+) currents in a concentration-dependent manner with an IC(50) of 17 microM. The high sensitivity of T-type Ca(2+) channels to nickel blockade combined with sequencing of a partial cDNA suggests that T-type Ca(2+) currents are generated by alpha1H subunits in chick nodose neurons. Steady-state activation and inactivation kinetics were similar to those previously reported for other alpha1H channels in mammalian neurons. Semi-quantitative PCR analysis indicates that alpha1H mRNA was present in chick nodose neurons by E7, suggesting that the functional expression of T-type Ca(2+) channels involves a posttranscriptional mechanism. These findings demonstrate a distinct pattern of T-type Ca(2+) channel functional expression in placode-derived neurons when compared with CNS neurons.

Extrinsic regulation of T-type Ca(2+) channel expression in chick nodose ganglion neurons.

Functional expression of T-type Ca(2+) channels is developmentally regulated in chick nodose neurons. In this study we have tested the hypothesis that extrinsic factors regulate the expression of T-type Ca(2+) channels in vitro. Voltage-gated Ca(2+) currents were measured using whole-cell patch clamp recordings in E7 nodose neurons cultured under various conditions. Culture of E7 nodose neurons for 48 h with a heart extract induced the expression of T-type Ca(2+) channels without any significant effect on HVA currents. T-type Ca(2+) channel expression was not stimulated by survival promoting factors such as BDNF. The stimulatory effect of heart extract was mediated by a heat-labile, trypsin-sensitive factor. Various hematopoietic cytokines including CNTF and LIF mimic the stimulatory effect of heart extract on T-type Ca(2+) channel expression. The stimulatory effect of heart extract and CNTF requires at least 12 h continuous exposure to reach maximal expression and is not altered by culture of nodose neurons with the protein synthesis inhibitor anisomycin, suggesting that T-type Ca(2+) channel expression is regulated by a posttranslational mechanism. Disruption of the Golgi apparatus with brefeldin-A inhibits the stimulatory effect of heart extract and CNTF suggesting that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Heart extract- or CNTF-evoked stimulation of T-type Ca(2+) channel expression is blocked by the Jak/STAT and MAP kinase blockers, AG490 and U0126, respectively. This study provides new insights into the electrical differentiation of placode-derived sensory neurons and the role of extrinsic factors in regulating the functional expression of Ca(2+) channels.