Craig H. Gelband

Alterations in rat interlobar artery membrane potential and K+ channels in genetic and nongenetic hypertension

The renal vasculature plays an important role in the control of blood pressure. K+ channels have been demonstrated to regulate smooth muscle membrane potential and thereby control smooth muscle tone. However, few data are available on K+ channel function in the renal vasculature of hypertensive animals. This study details changes in K+ currents and membrane potential in genetic and nongenetic models of hypertension. The patch-clamp technique and Ca(2+)-imaging fluorescence were used to examine the differences in Wistar-Kyoto (WKY), Sprague-Dawley (SD), spontaneously hypertensive (SHR), and deoxycorticosterone acetate (DOCA) hypertensive single cells of rat kidney interlobar arteries. In current-clamp experiments, SHR and DOCA hypertensive cells were approximately 20 mV more depolarized than the control cells. In voltage-clamp experiments with 4-amino-pyridine and niflumic acid present to inhibit voltage-dependent K+ (K(v)) and Ca(2+)-activated CI- (CI(Ca)) currents, SHR and DOCA hypertensive Ca(2+)-activated K+ (K(Ca)) currents were significantly larger and activated at more negative potentials than the control. Conversely, with charybdotoxin and niflumic acid present to inhibit K(Ca) and CI(Ca) currents, SHR and DOCA hypertensive K(v) current was significantly smaller than the control. Finally, basal and angiotensin II-stimulated peak intracellular free [Ca2+] was greater in the SHR and DOCA hypertensive cells compared with control cells. These results suggest that membrane potential and the activity of K(Ca) and K(v) channels are altered in hypertensive rat renal interlobar arteries and may play a role in the regulation of renal blood flow under physiological and patho-physiological conditions.

Ca2+ Release From Intracellular Stores Is an Initial Step in Hypoxic Pulmonary Vasoconstriction of Rat Pulmonary Artery Resistance Vessels

BACKGROUND A reduction in oxygen tension in the lungs is believed to inhibit a voltage-dependent K+ (Kv) current, which is thought to result in membrane depolarization leading to hypoxic pulmonary vasoconstriction (HPV). However, the direct mechanism by which hypoxia inhibits Kv current is not understood. METHODS AND RESULTS Experiments were performed on rat pulmonary artery resistance vessels and single smooth muscle cells isolated from these vessels to examine the role of Ca2+ release from intracellular stores in initiating HPV. In contractile experiments, hypoxic challenge of endothelium-denuded rat pulmonary artery resistance vessels caused either a sustained or transient contraction in Ca2+-containing or Ca2+-free solution, respectively (n=44 vessels from 11 animals). When the ring segments were treated with either thapsigargin (5 micromol/L), ryanodine (5 micromol/L), or cyclopiazonic acid (5 micromol/L) in Ca2+-containing or Ca2+-free solution, a significant increase in pulmonary arterial tone was observed (n=44 vessels from 11 animals). Subsequent hypoxic challenge in the presence of each agent produced no further increase in tone (n=44 vessels from 11 animals). In isolated pulmonary resistance artery cells loaded with fura 2, hypoxic challenge, thapsigargin, ryanodine, and cyclopiazonic acid resulted in a significant increase in [Ca2+]i (n=18 cells from 6 animals) and depolarization of the resting membrane potential (n=22 cells from 6 animals). However, with prior application of thapsigargin, ryanodine, or cyclopiazonic acid, a hypoxic challenge produced no further change in [Ca2+]i (n=18 from 6 animals) or membrane potential (n=22 from 6 animals). Finally, application of an anti-Kv1.5 antibody increased [Ca2+]i and caused membrane depolarization. Subsequent hypoxic challenge resulted in a further increase in [Ca2+]i with no effect on membrane potential (n=16 cells from 4 animals). CONCLUSIONS In rat pulmonary artery resistance vessels, an initial event in HPV is a release of Ca2+ from intracellular stores. This rise in [Ca2+]i causes inhibition of voltage-dependent K+ channels (possibly Kv1.5), membrane depolarization, and an increase in pulmonary artery tone.

Angiotensin receptors and norepinephrine neuromodulation: implications of functional coupling.

The objective of this review is to examine the role of neuronal angiotensin II (Ang II) receptors in vitro. Two types of G protein-coupled Ang II receptors have been identified in cardiovascularly relevant areas of the brain: the AT1 and the AT2. We have utilized neurons in culture to study the signaling mechanisms of AT1 and AT2 receptors. Neuronal AT1 receptors are involved in norepinephrine (NE) neuromodulation. NE neuromodulation can be either evoked or enhanced. Evoked NE neuromodulation involves AT1 receptor-mediated, losartan-dependent, rapid NE release, inhibition of K+ channels and stimulation of Ca2+ channels. AT1 receptor-mediated enhanced NE neuromodulation involves the Ras-Raf-MAP kinase cascade and ultimately leads to an increase in NE transporter, tyrosine hydroxylase and dopamine beta-hydroxylase mRNA transcription. Neuronal AT2 receptors signal via a Gi protein and are coupled to activation of PP2A and PLA2 and stimulation of K+ channels. Finally, putative cross-talk pathways between AT1 and AT2 receptors will be discussed.

Angiotensin IV receptor-mediated activation of lung endothelial NOS is associated with vasorelaxation.

The hexapeptide angiotensin (ANG) IV, a metabolic product of ANG II, has been reported to play a functional role in the regulation of blood flow in extrapulmonary tissues. Here, we demonstrate that ANG IV-specific (AT4) receptors are present in porcine pulmonary arterial endothelial cells (PAECs) and that the binding of ANG IV to AT4 receptors can be blocked by its antagonist divalinal ANG IV but not by the ANG II-, AT1-, and AT2-receptor blockers [Sar1,Ile8]ANG II, losartan, and PD-123177, respectively. ANG IV significantly increased endothelial cell constitutive nitric oxide synthase (ecNOS) activity ( P < 0.05) as well as cellular cGMP content ( P < 0.001). Western blot analysis revealed that ecNOS protein expression was comparable in control and ANG IV-stimulated cells. Divalinal ANG IV but not [Sar1,Ile8]ANG II, losartan, or PD-123177 inhibited the ANG II- and ANG IV-stimulated increases in ecNOS activity and cGMP content in PAECs. Incubation in the presence of N-nitro-l-arginine methyl ester (l-NAME) or methylene blue but not of indomethacin significantly diminished ANG IV-stimulated as well as basal levels of cGMP ( P < 0.001). Similarly, in situ studies with precontracted porcine pulmonary arterial rings showed that ANG IV caused an endothelium-dependent relaxation that was blocked byl-NAME or methylene blue. Collectively, these results demonstrate that ANG IV binds to AT4 receptors, activates ecNOS by posttranscriptional modulation, stimulates cGMP accumulation in PAECs, and causes pulmonary arterial vasodilation, suggesting that ANG IV plays a role in the regulation of blood flow in the pulmonary circulation.

Functional Interactions Between Neuronal AT1 and AT2 Receptors

Angiotensin II (Ang II), via the activation of the AT1 and AT2 receptors regulates electrophysiological responses of catecholaminergic neurons. This study was designed to determine if functional interactions between AT1 and AT2 receptors exist in a single neuron. Ang II caused two unique electrophysiological responses characteristic of receptor crosstalk. First, Ang II elicited an AT1 receptor-mediated decrease in IK followed by an AT2 receptor-mediated increase in IK. Second, Ang II elicited an AT2 receptor-mediated increase in IK followed by an AT1 receptor-mediated decrease in IK. AT1 and AT2 receptors were co-localized on the catecholaminergic neurons. These observations suggest, for the first time, the existence of a crosstalk between Ang II receptor subtypes that may be significant in the physiological activity of catecholaminergic neurons.

Ion Channels in Vascular Smooth Muscle: Alterations in Essential Hypertension

Abstract Essential hypertension is characterized by a near normal cardiac output but an increase in total peripheral resistance. In turn, total peripheral resistance is controlled directly by the diameter of the small arteries and arterioles like those in the kidney. The dynamic regulation of renal vessel diameter is governed by the contractile state of the vascular smooth muscle cells that line the vessel walls. This review addresses the role of a number of different ion channels to initiate and maintain the contractile state of the vascular smooth muscle cells in hypertension and the potential prevention of hypertension through gene therapy. These specific channels include Ca2+, KCa, Kv, and Cl~ channels. In hypertension, it has been reported that increased activity of Ca2+ channels and decreased activity of Kv channels are responsible for the increased contractile tone and resting membrane potential observed in dissociated vascular smooth muscle cells from the spontaneously hypertensive rat. In contrast, increased activity of KCa channels in vascular smooth muscle cells of the SHR has been hypothesized to dampen or brake the activity of Ca2+ and Kv channels. Finally, recent evidence suggests that introducing angiotensin II type-1 receptor antisense into prehypertensive rat pups prevents the onset of pathophysiological alterations observed in hypertension including K+ channel alterations. These results suggest that gene therapy may be a useful pharmacological and physiological tool to combat hypertension.

Angiotensin II type 1 receptor-modulated signaling pathways in neurons

Mammalian brain contains high densities of angiotensin II (Ang II) type 1 (AT1) receptors, localized mainly to specific nuclei within the hypothalamus and brainstem regions. Neuronal AT1 receptors within these areas mediate the stimulatory actions of central Ang II on blood pressure, water and sodium intake, and vasopressin, secretion, effects that involve the modulation of brain noradrenergic pathways. This review focuses on the intracellular events that mediate the functional effects of Ang II in neurons, via AT1 receptors. The signaling pathways involved in shortterm changes in neuronal activity, membrane ionic currents, norepinephrine (NE) release, and longer-term neuromodulatory actions of Ang II, are discussed. It will be apparent from this discussion that the signaling pathways involved in these events, are often distinct.

Reversal of hypertension by angiotensin II type 1 receptor antisense gene therapy in the adult SHR

Pharmacological blockade of the renin-angiotensin system in both hypertensive patients and animal models such as the spontaneously hypertensive rat (SHR) effectively reduces blood pressure (BP). Recent studies have established that virally mediated delivery (vector LNSV) of antisense to the angiotensin II type 1 receptor (LNSV-AT1R-AS) will attenuate or abolish the development of hypertension in the SHR. However, the effectiveness of this gene therapy approach to reduce high BP once it is established in the adult has not been ascertained. In this study, we investigated the hypothesis that viral delivery of AT1R-AS into the adult SHR will reduce BP and reverse the vascular reactivity associated with the hypertension. Intracardiac injection of virus particles containing LNSV-AT1R-AS into adult SHR resulted in a 30- to 60-mmHg reduction in BP that was maintained for up to 36 days compared with SHR treated with virus alone (LNSV without antisense). Measurement of renal resistance arteriolar reactivity demonstrated a leftward shift in the KCl and phenylephrine concentration-response relationships and an impaired endothelium-dependent relaxation to ACh in LNSV-treated SHR compared with control Wistar-Kyoto rats. These vascular alterations were reversed in the LNSV-AT1R-AS-treated SHR. Collectively, these data demonstrate that virally mediated gene delivery of AT1R-AS can effectively reduce BP and reverse renovascular pathophysiology associated with the hypertensive state when administered to the adult SHR.

Angiotensin II Type 1 Receptor–Mediated Inhibition of K+ Channel Subunit Kv2.2 in Brain Stem and Hypothalamic Neurons

Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT1) receptor-mediated inhibition of total outward K+ current that contributes to an increase in neuronal firing rate. However, the exact K+ conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K+ current revealed a component of K+ current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC50 values of 21.7 micromol/L, 1.49 mmol/L, and 890 micromol/L, respectively, and insensitive to alpha-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 micromol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K+ current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT2 receptor blocker PD123,319, elicited an inhibition of neuronal K+ current that was abolished by quinine (50 micromol/L). Reverse transcriptase-polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT1 receptor in Xenopus oocytes demonstrated an Ang II-induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT1 receptor-mediated reduction of neuronal K+ current and subsequently to the modulation of cardiovascular function.

Targeting of the renin-angiotensin system by antisense gene therapy: a possible strategy for the long-term control of hypertension.

Traditional pharmacological agents have been successfully used for the treatment of hypertension for a number of decades. However, this therapeutic regimen has reached a conceptual plateau and a cure for the disease is far from appearing on the horizon. With this in mind, and recent advances in state of the art gene delivery system coupled with the anticipated completion of the human genome project, it is timely to think about the possibility of treating and/or curing hypertension using genetic means. In this review, we discuss the role of renin–angiotensin system (RAS) in hypertension; the current gene delivery/gene transfer systems and the RAS as a target for gene therapy to treat hypertension; the successful use of retroviral vectors to deliver antisense to the AT1 receptor (AT1-AS) to prevent the development of hypertension and cardiovascular pathophysiology; the potential use of the viral vectors for the reversal of hypertension; and the future of antisense gene therapy and potential advantages and limitations of this regimen in the treatment and/or control of hypertension.