James P. Porter

A comparison of the hemodynamic effects produced by electrical stimulation of subnuclei of the paraventricular nucleus

The paraventricular nucleus of the hypothalamus (PVN) is composed of magnocellular and parvocellular subdivisions. Magnocellular neurosecretory neurons project to the neurohypophysis while parvocellular neurons send monosynaptic axonal projections to autonomic regulatory areas in the brainstem and spinal cord. In the present study, we investigated the hemodynamic effects produced by selective magnocellular or parvocellular stimulation. In urethane anesthetized rats with intact baroreflexes, magnocellular and parvocellular stimulation produced only slight differences in hemodynamic responses, however, following acute sinoaortic denervation a clear difference was observed. Parvocellular stimulation produced an increase in arterial pressure and vasoconstriction in gut, kidney and skeletal muscle. Magnocellular stimulation produced little effect on arterial pressure and marked vasodilation in the hindquarters. Blockade of peripheral vasopressin vascular receptors did not affect the vasoconstrictor response produced by stimulation of PVN. These data are consistent with the hypothesis that the long descending neural projections of the parvocellular PVN subserve a selective vasoconstrictor function.

Role of Cytoplasmic Tail of the Type 1A Angiotensin II Receptor in Agonist– and Phorbol Ester–Induced Desensitization

To investigate mechanisms underlying the agonist-induced desensitization of the type 1A angiotensin II receptor (AT1A-R), we have stably expressed in Chinese hamster ovary (CHO) cells the wild-type receptor and truncated mutants lacking varying lengths of the cytoplasmic tail. Assay of inositol 1,4,5-trisphosphate (IP3) formation in response to agonist demonstrated that the truncated mutants T318, T328, and T348 lacking the last 42, 32, or 12 amino acid residues, respectively, couple with Gq protein with an efficiency similar to that of full-length receptors, whereas coupling of Gq protein was abolished in the T310 truncated mutant devoid of the carboxyl-terminal 50 amino acids. Exposure of CHO/AT1A-R cells expressing the wild-type AT1A-R to angiotensin II resulted in rapid and dose-dependent homologous desensitization of receptor-mediated IP3 formation, which was independent of the receptor internalization. Mastoparan, an activator of G protein-coupled receptor kinase (GRK), induced desensitization of the AT1A-R. The agonist-induced desensitization of the receptor was largely prevented by heparin, a potent inhibitor of GRK, whereas it was only partially attenuated by a protein kinase C (PKC)-specific inhibitor. The homologous or heterologous desensitization of the receptor was greatly impaired in the truncated mutants T318 and T328, lacking the Ser/Thr-rich (13 or 12 Ser/Thr residues) cytoplasmic tail of the AT1A-R. Deletion of the last two Ser residues, including one PKC consensus site in the receptor tail, prevented only phorbol 12-myristate 13-acetate-induced desensitization by 30%. Moreover, we found an agonist-induced translocation of a heparin-sensitive kinase activity. The angiotensin II-stimulated heparin-sensitive kinase could phosphorylate a thioredoxin fusion protein containing the entire AT1A-R cytoplasmic tail (N295 to E359), which lacks consensus phosphorylation sites for GRK1, GRK2, and GRK3. The heparin-sensitive kinase may not be GRK2, GRK3, or GRK6 expressed in CHO/AT1A-R cells, since angiotensin II did not induce translocation of these receptor kinases. Potential Ser/Thr phosphorylation sites located between S328 and S347 in the cytoplasmic tail of AT1A-R seem to play a critical role in the heterologous and homologous desensitization of the receptor. A heparin-sensitive kinase other than GRK2, GRK3, or GRK6 may be involved in the agonist-induced homologous desensitization of the AT1A-R.

Functional role of brain AT1 and AT2 receptors in the central angiotensin II pressor response

Intracerebroventricular (i.c.v.) angiotensin II (ANG II) increases vascular resistance and elicits a pressor response characterized by sympathetic nervous system activation (SNS component) and increased vasopressin (VP) secretion (VP component). This study examines the role of brain AT1 and AT2 ANG II receptors in mediating the pressor and renal hemodynamic effects of i.c.v. ANG II in conscious Sprague-Dawley rats. Mean arterial pressure, heart rate and renal vascular resistance responses to i.c.v. ANG II (100 ng in 5 microliters) were determined 10 min after i.c.v. injection of either the AT1 receptor antagonist, DuP 753 (1.0, 2.5, 5.0, 10.0 micrograms), the AT2 receptor ligand, PD 123319 (3.5 x [10(-6), 10(-4), 10(-2), 10(0)] micrograms), or both. In control rats, i.c.v. DuP 753 prevented the pressor response and the increase in renal vascular resistance that occurred following i.c.v. ANG II in a dose-dependent manner (P < 0.05), while i.c.v. PD 123319 was without affect. When the VP- and SNS components were studied individually, by preventing the SNS component with intravenous (i.v.) chlorisondamine or the VP component with a V1 receptor antagonist (i.v.) similar results were obtained; DuP 753 prevented the SNS component and significantly reduced the VP component. These results indicate that both central ANG II pressor components are mediated primarily by brain AT1 receptors. However, doses of DuP 753 were more effective when combined with 3.5 micrograms of PD 123319 than when given alone (P < 0.05), suggesting that the pressor effects of i.c.v. ANG II may involve activation of multiple ANG II receptor subtypes.

Effect of central angiotensin II on body weight gain in young rats

Systemic infusion of ANG II decreases body weight and food intake and increases energy expenditure. We recently reported that young rats receiving a 1-week intracerebroventricular (i.c.v.) infusion of angiotensin II (ANG II) exhibited decreased body weight compared to control. The aim of the present investigation was to determine if chronic i.c.v. infusion of ANG II also decreases food intake and increases energy expenditure. Young rats were infused with i.c.v. 0.9% saline or ANG II (16.7 or 4.2 ng/min) for at least 10 days and body weight and food intake were monitored daily. Pair-fed rats had the same daily food intake as the ANG II-infused rats. The i.c.v. ANG II decreased body weight gain and food intake. The decrease in weight gain was greater than in the pair-fed groups. The expression of mRNA for uncoupling protein-1 (UCP-1) in BAT was increased significantly in the ANG II-infused rats compared to the pair-fed animals. Subcutaneous infusion of ANG II at the same doses used for i.c.v. infusion had no effect on body weight or food intake. The expression of CRH mRNA in the paraventricular nucleus was not increased in the ANG II-infused rats. These data are consistent with the idea that i.c.v. ANG II decreases body weight gain in young rats, in part, by decreasing food intake and, in part, by increasing thermogenesis (although via a CRH-independent mechanism). This central effect of ANG II may contribute to or complement the effect of peripheral ANG II on body weight.

The disposition of dapsone in cirrhosis

Acetylation and N‐hydroxylation of dapsone were evaluated in drug‐free, non‐smoking, normal subjects and subjects with cirrhosis (n = 7 for each group) after oral administration of 100 mg dapsone. Acetylation was not correlated with oral dapsone clearance or reduced in cirrhosis (0.37 ± 0.43 versus 0.52 ± 0.32). Fractional metabolic clearance of dapsone to its hydroxylamine was associated with dapsone oral clearance (r = 0.96, p < 0.001, n = 14). In patients with cirrhosis, liver disease was associated with a trend to reduction in oral clearance (22%) and metabolic clearance of dapsone (48%). Protein binding was minimally reduced by cirrhosis (73% ± 1% versus 69% ± 3% in patients with cirrhosis (p < 0.02). The dapsone recovery ratio was validated as a phenotypic index of the metabolic clearance of dapsone (r = 0.74, p < 0.05). In an extended comparison of 14 patients with cirrhosis to 70 control subjects, cirrhosis was associated with reductions of 28% in dapsone recovery ratio (p < 0.001), and 37% in acetylation ratio (p < 0.01). Neither dapsone recovery ratio nor acetylation ratio correlated with Pugh Score, conventional liver function tests, indocyanine green clearance, or phenotypic measures of S‐mephenytoin hydroxylase or debrisoquin hydroxylase activity. We conclude that cirrhosis is associated with minor changes in dapsone disposition and that dosage modification is not required. In addition, there is evidence that cirrhosis has a selective influence on activity of individual isozymes of cytochrome P450.

Functional roles of brain AT1 and AT2 receptors in the central angiotensin II pressor response in conscious young spontaneously hypertensive rats

Areas of adult rat brain that mediate the cardiovascular effects of central angiotensin II (ANG II) predominantly express AT1 ANG II receptors. In contrast, AT2 receptor expression in young rats is transiently increased, reaching a maximum during the first few weeks of life. This study was designed to determine the roles of brain AT1 and AT2 receptors in mediating the central pressor effects of ANG II in young (4-week-old) conscious spontaneously hypertensive rats (SHR). Mean arterial pressure responses to intracerebroventricular (i.c.v.) ANG II (100 ng in 5 microliters) were determined 10 minutes after i.c.v. injection of either the AT1 receptor antagonist Losartan (1.0, 2.5, 5.0, and 10.0 micrograms), the AT2 receptor ligand PD 123319 (3.5 x [10(-6), 10(-4), 10(-2), 10(0)] micrograms), or both. In control rats, i.c.v. Losartan prevented the pressor response to i.c.v. ANG II in a dose-dependent manner (P < 0.05), while i.c.v. PD 123319 alone was without effect. In other animals, pressor responses caused by i.c.v. ANG II-induced vasopressin secretion (VP-component) and sympathetic nervous system activation (SNS-component) were studied individually, with similar result; Losartan prevented the SNS-component, but reduced the VP-component by only 45%, indicating that both pressor components involve AT1 receptor activation. However, doses of Losartan were more effective when combined with 3.5 micrograms of PD 123319 than when given alone (P < 0.05); nearly eliminating the VP-component. These results suggest that i.c.v. ANG-II-induced pressor effects may involve activation of multiple receptor subtypes.

Ceramides mediate cigarette smoke-induced metabolic disruption in mice

Cigarette smoke exposure increases lung ceramide biosynthesis and alters metabolic function. We hypothesized that ceramides are released from the lung during cigarette smoke exposure and result in elevated skeletal muscle ceramide levels, resulting in insulin resistance and altered mitochondrial respiration. Employing cell and animal models, we explored the effect of cigarette smoke on muscle cell insulin signaling and mitochondrial respiration. Muscle cells were treated with conditioned medium from cigarette smoke extract (CSE)-exposed lung cells, followed by analysis of ceramides and assessment of insulin signaling and mitochondrial function. Mice were exposed to daily cigarette smoke and a high-fat, high-sugar (HFHS) diet with myriocin injections to inhibit ceramide synthesis. Comparisons were conducted between these mice and control animals on standard diets in the absence of smoke exposure and myriocin injections. Muscle cells treated with CSE-exposed conditioned medium were completely unresponsive to insulin stimulation, and mitochondrial respiration was severely blunted. These effects were mitigated when lung cells were treated with the ceramide inhibitor myriocin prior to and during CSE exposure. In mice, daily cigarette smoke exposure and HFHS diet resulted in insulin resistance, which correlated with elevated ceramides. Although myriocin injection was protective against insulin resistance with either smoke or HFHS, it was insufficient to prevent insulin resistance with combined CS and HFHS. However, myriocin injection restored muscle mitochondrial respiration in all treatments. Ceramide inhibition prevents metabolic disruption in muscle cells with smoke exposure and may explain whole body insulin resistance and mitochondrial dysfunction in vivo.

Chronic intracerebroventricular infusion of angiotensin II increases brain AT1 receptor expression in young rats

The aim of the present study was to determine the effect of chronic intracerebroventricular infusion of angiotensin II (ANG II) on the expression of brain AT1 receptors in young (3-4 weeks) rats. One week of icv ANG II infusion produced a significant increase in brain AT1 receptor protein (Western blot) and mRNA (relative RT-PCR) expression. These data raise the possibility that ANG II may play a role in postnatal expression of brain AT1 receptors.

Role of Paraventricular and Arcuate Nuclei in Cardiovascular Regulation

There has been an increasing awareness in recent years of the potential for structures in forebrain to influence the cardiovascular system. Much attention has been focused on the paraventricular nucleus, which has been defined neuroanatomically to contain both magnocellular units that project to the neurohypophysis and parvocellular neurons that descend monosynaptically to the intermediolateral column of the spinal cord. Interest in the paraventricular nucleus has also extended to its role as a source of both humorally derived vasopressin and of descending projections from parvocellular regions of the nucleus that contain separate vasopressin and oxytocin-containing fibers. Portions of the studies reviewed in this chapter describe experiments designed to evaluate the role of the parvocellular paraventricular nucleus in cardiovascular regulation and to determine whether vasopressinergic projections to the spinal cord contribute to cardiovascular effects produced by excitation of this region.

Phytoestrogens: Implications in Neurovascular Research

The early discontinuation of the Womens Health Initiative trial evaluating the effects of estrogen plus progestin due to concerns about the risk-benefit ratio of this steroid combination therapy emphasizes the need to examine alternative methods of estrogen replacement. One such alternative is isoflavone consumption of soy-derived dietary phytoestrogens that have received prevalent usage due to their ability to decrease age related disease (cardiovascular and osteoporosis), hormone-dependent cancers (breast and prostate), and peri- and postmenopausal symptoms. Differences in dietary phytoestrogen consumption result in large variations in somatic phytoestrogen content. These molecules affect estrogen and estrogen receptor function in several ways, including having both agonist and antagonist effects on estrogen receptors, as well as functioning like natural selective estrogen receptor modulators. Similar to estrogens, dietary phytoestrogens appear to affect certain aspects of vascular, neuroendocrine, and cognitive function. This article reviews health effects of estrogen, isoflavones and their hormonal mechanism of action, brain penetration by isoflavones, heath effects of isoflavones, and effects of isoflavones on vascular, neuroendocrine, and cognitive function. Because of their diverse health effects and widespread availability in soy foods, dietary phytoestrogens merit continued research into their effects on human health and cognitive function.