Dopamine and norepinephrine responses to film-induced sexual arousal in sexually functional and sexually dysfunctional women.

This study was designed to assess potential differences between sexually functional and dysfunctional women in dopamine (DA) and norepinephrine (NE) responses to erotic stimuli. Blood levels of homovanillic acid (HVA; the major metabolite of DA) and NE were taken during the showing of a nonsexual and a sexual film from 9 women with female sexual arousal disorder and hypoactive sexual desire disorder and from 13 sexually functional women. We assessed sexual arousal subjectively using a self-report scale and physiologically using a vaginal photoplethysmograph. HVA levels significantly decreased in sexually functional and dysfunctional women during the erotic versus during the neutral film. NE levels were not significantly different for either group of women during the neutral and erotic films. Sexually dysfunctional women had significantly higher levels of NE during both the neutral and erotic films compared with functional women. Subjective or physiological arousal differences between neutral and erotic films were not significantly different between functional and dysfunctional women.

Glucagon stimulation of hepatic Na(+)-pump activity and alpha-subunit phosphorylation in rat hepatocytes.

In this study the possible role of Na+ influx, arachidonate mediators and alpha-subunit phosphorylation in the stimulatory response of hepatic Na+/K(+)-ATPase to glucagon was examined. Glucagon stimulation of ouabain-sensitive 86Rb+ uptake in freshly isolated rat hepatocytes reached maximal levels in less than 1 min after hormone addition and was half-maximal (EC50) at a concentration of 2.4( +/- 1.3) x 10(-10) M. Analysis of the K(+)-dependence of this response indicates an effect on the apparent Vmax. for K+ with no significant change in the apparent kappa 0.5. Unlike monensin, glucagon stimulation of Na+/K(+)-ATPase-mediated transport activity was not associated with an increase in 22Na+ influx. This indicates that the stimulation of Na+/K(+)-ATPase by glucagon is not secondary to an increase in Na+ influx. A role for arachidonate mediators in this effect also appears unlikely because neither basal nor glucagon-stimulated ouabain-sensitive 86Rb+ uptake was significantly affected by supramaximal concentrations of cyclo-oxygenase, lipoxygenase, cytochrome p-450 or phospholipase A2 inhibitors. To study the possible role of protein kinase-mediated phosphorylation in the stimulation of ouabain-sensitive 86Rb uptake, hepatocytes were metabolically radiolabelled with [32P]P(i), Glucagon stimulated incorporation of 32P into a 95 kDa phosphoprotein that comigrates with Na+/K(+)-ATPase alpha-subunit immunoreactivity in two-dimensional gel electrophoresis. The alpha-subunit could be immunoprecipitated from detergent-solubilized particulate fractions of hepatocytes using an anti-(rat kidney Na+/K(+)-ATPase) serum. When hepatocytes were metabolically radiolabelled with [32P]P(i), the immunoprecipitated alpha-subunit contained 32P. Glucagon increased the incorporation of 32P into the immunoprecipitated subunit by 197 +/- 21% (n = 6). Similar results were observed with a rabbit anti-peptide serum ('anti-LEAVE' serum) prepared against an amino acid sequence in the alpha-subunit. The EC50 for glucagon-stimulated phosphorylation of the alpha-subunit (approximately 1 x 10(-10) M) was very close to that for glucagon stimulation of ouabain-sensitive 86Rb+ uptake. In conclusion, it appears that glucagon stimulation of hepatic Na+/K(+)-ATPase-mediated transport activity is not secondary to increases in Na+ influx or changes in the levels of an arachidonate mediator. The data provide support for the hypothesis that glucagon stimulation of Na(+)-pump activity in hepatocytes may be related to protein kinase-mediated changes in the phosphorylation state of the alpha-subunit.

Okadaic acid stimulates ouabain-sensitive 86Rb(+)-uptake and phosphorylation of the Na+/K(+)-ATPase alpha-subunit in rat hepatocytes.

Ca(2+)-mobilizing and cAMP-dependent hormones rapidly increase sodium, potassium-dependent adenosine triphosphatase (Na+/K(+)-ATPase)-mediated transport in rat hepatocytes. To explore the possible role of protein phosphatases in these responses we used a protein phosphatase inhibitor, okadaic acid. Okadaic acid stimulation of ouabain-sensitive 86Rb(+)-uptake was maximal between two and three minutes and displayed an EC50 of 41 +/- 1 nM. Inhibition of Na+/H+ exchange with an amiloride analog abolished the response to insulin, but had no effect on okadaic acid-mediated stimulation of Na+/K(+)-ATPase transport. In hepatocytes metabolically-radiolabeled with 32Pi, okadaic acid stimulated the incorporation of radioactivity into several 95 kDa peptides, one of which reacted with anti-LEAVE peptide antisera, that recognizes Na+/K(+)-ATPase alpha-subunits. In other experiments Na+/K(+)-ATPase was immunoprecipitated from detergent-solubilized membrane fractions of metabolically-radiolabeled cells with an antisera to purified rat kidney Na+/K(+)-ATPase. A 95 kDa phosphoprotein was immunoprecipitated using anti-Na+/K(+)-ATPase antisera, but not by preimmune serum. Okadaic acid stimulated incorporation of radioactivity into this band by 220 +/- 28%. These findings provide support for the hypothesis that rapid stimulation of hepatic Na+/K(+)-ATPase by hormones may be related to protein kinase/phosphatase-mediated changes in the phosphorylation state of the Na+/K(+)-ATPase alpha-subunit.

Altered expression of skeletal muscle proteins during sepsis.

Sepsis induces a net catabolic state in gastrocnemius by increasing protein degradation and decreasing protein synthesis. To determine whether or not sepsis induces a preferential effect on the expression of individual proteins, proteins from gastrocnemius muscle of control and septic rats were separated by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laser densitometry of proteins stained with silver provided evidence that the relative abundance of thirty-five proteins was significantly (p < .05) and reproducibly increased during sepsis compared to control. No individual protein underwent significant down-regulation in their relative abundance during sepsis. Twenty-three of the 35 proteins identified in two-dimensional gels of the gastrocnemius were also present in the plasma of septic rats. The remaining 12 proteins, therefore, were taken to represent skeletal muscle proteins. One of the 12 proteins was identified by immunoblot analysis to be carbonic anhydrase III. Another of the proteins was identified as triosephosphate isomerase based upon microsequencing of the N terminus.

Pyruvate carboxylase in genetic obesity.

Immunoblotting and protein microsequencing were used to identify several adipocyte proteins expressed in an obesity-related fashion in the Zucker rat. One of these was a 116-kDa particulate protein (p116). The p116 levels in adipocytes from 5- to 7-wk-old obese Zucker rats were two- to fivefold higher on a per milligram of protein basis than levels in lean animals and decreased after the induction of streptozotocin-induced diabetes mellitus. This suggests the change may be related to the actions of insulin. Hepatic levels of p116 did not change. The p116 was purified to homogeneity from obese Zucker rat adipocytes, and polyclonal antisera were prepared against the purified protein in rabbits. Microanalysis of electroblotted p116 proteolytic fragments suggested that p116 was pyruvate carboxylase (PC). Other evidence that p116 was PC included the following: 1) p116 contained biotin, 2) p116 in particulate subcellular fractions was soluble after freeze-lysis, 3) antibodies to p116 reacted with purified hepatic PC, 4) p116 and purified hepatic PC had identical pI and relative molecular weight values, and 5) similar changes were detected in adipocyte p116 and PC enzyme activity during obesity and after the induction of streptozotocin-induced diabetes mellitus. Increased adipose tissue PC probably contributes to the increased lipogenic capacity of young obese Zucker rat adipocytes.