Grant G. Kelley

Protein phosphorylation changes in bovine carotid artery smooth muscle during contraction and relaxation

The protein phosphorylation changes associated with the contraction and relaxation of bovine carotid artery smooth muscle were studied using two-dimensional gel electrophoresis of labeled phosphoproteins. Muscle was stimulated with histamine, angiotensin II, 12-deoxyphorbol 13-isobutyrate (DPB) or high extracellular K+. Histamine induced a rapid and sustained contraction which was associated with an early (2 min) phosphorylation of 20 kDa myosin light chain (MLC) and two cytosolic proteins, Nos. 1 and 2, and with the late (60 min) phosphorylation of MLC, two isoelectric variants of desmin and ten other cytosolic proteins. Additionally, there was a decrease in the extent of phosphorylation of two cytosolic proteins, Nos. 9 and 10. Angiotensin II induced a rapid but transient contraction which was associated with the same early (2 min) phosphorylation changes, but with none of the late (60 min) changes. Elevation of the extracellular K+ concentration to 110 mM led to a sustained contraction which was associated with the phosphorylation of MLC and proteins Nos. 1 and 2 at both 2 and 60 min, but none of the other late phase phosphoproteins were seen. Addition of DPB, an activator of protein kinase C, induced a slowly developing but sustained contractile response which was associated with none of the early (5 min) phosphorylation changes. However, nearly all of late (60 min) protein phosphorylation changes were the same as those seen after histamine action. Addition of forskolin to either control or histamine-treated muscle led to an increase in the phosphorylation of three cytosolic proteins (Nos. 3, 8 and 13), and in the histamine-contracted muscle the dephosphorylation of MLC and proteins Nos. 4, 9, 10, 15 and 16. Similarly, forskolin induced a relaxation of DPB-treated muscle and the dephosphorylation of proteins Nos. 4, 9, 10, 15 and 16. These results suggest that there are two pathways by which histamine activates contraction: a Ca2+-calmodulin pathway which initiates the response, and a protein kinase C pathway which, along with the Ca2+-calmodulin pathway, sustains contraction.

Time-dependent effects of cholinergic stimulation on beta cell responsiveness

The effects of cholinergic stimulation on beta cell insulin secretory and phosphoinositide (PI) responses were determined in freshly isolated rat islets. Increasing the glucose level perifusing the islet from 5.6 to 8 mM was accompanied by a modest insulin secretory response. The further addition of 10 μM carbachol increased peak first- and second-phase responses by 2.6- and 6.8-fold, respectively. In the presence of 5.6 mM glucose, this low level (10 μM) of carbachol increased insulin release two-to three-fold, a response that was maintained for at least 60 min. In contrast to these acute stimulatory actions in the presence of glucose, chronic 3.5-h exposure of islets to 10 μM carbachol abolished beta cell insulin secretory responses to stimulation, with the combination of 8 mM glucose plus 10 μM carbachol. However, the further addition of 200 μM tolbutamide to these islets increased insulin secretory rates significantly. To establish the role of islet cell PI hydrolysis in these secretory responses, additional studies were conducted with islets whose PI pools were labeled with [3H]inositol. Acute exposure to 10 μM carbachol alone significantly increased inositol phosphate accumulation and the efflux of [3H]inositol, even in the absence of glucose. Including 10 μM carbachol during the labeling period with [3H]inositol resulted in significant impairments in subsequently measured inositol phosphate accumulation and [3H]inositol efflux responses to 8 mM glucose plus carbachol stimulation. Prior long-term exposure to 10 μM carbachol also induced heterologous desensitization: 20 mM glucose-stimulated insulin release and inositol phosphate accumulation were impaired in a parallel fashion. Chronic carbachol exposure had no deleterious effect on the usage of 8 or 20 mM glucose or on the insulin content of the islet. The acute stimulatory effects of carbachol on inositol phosphate accumulation as well as its inhibitory effect on 20 mM glucose-stimulated insulin release after prolonged exposure to the muscarinic agonist were significantly reduced by atropine. These findings demonstrate that changes in PI hydrolysis parallel those observed with insulin secretion and suggest that alterations in phospholipase C activation may account, at least in part, for the insulin secretory responses observed.

Effects of short-term culturing on islet phosphoinositide and insulin secretory responses to glucose and carbachol

The ability of glucose and carbachol, alone or in combination, to stimulate islet cell phosphoinositide (PI) hydrolysis and insulin secretory responses in freshly isolated or in 20–24 h cultured rat islets was assessed. In freshly isolated,3H-inositol-prelabeled islets, 20 mM glucose alone or 1 mM carbachol alone stimulated significant increments in3H-inositol efflux and inositol phosphate (IP) accumulation. When stimulated with both agonists, a dramatic and synergistic effect on IP accumulation was noted. Carbachol (1 mM) alone had no sustained stimulatory effect on insulin secretion. Glucose (20 mM) alone induced a biphasic insulin secretory response. When compared to prestimulatory secretory rates of 18±4 pg/islet/min, peak first and second phase responses of freshly isolated islets to 20 mM glucose averaged 126±24 and 520±82 pg/islet/min, respectively. In the presence of both glucose (20 mM) and carbachol (1 mM), peak first and second phase responses now averaged 422±61 and 1016±156 pg/islet/min, respectively. In contrast to freshly studied islets, culturing islets for 20–24 h in CMRL-1066 medium attenuated all measured responses. The increases in3H-inositol efflux rates in response to glucose, carbachol, or their combination were significantly less than those observed with fresh islets. The IP responses were also attenuated. Second phase insulin secretory responses to 20 mM glucose alone (68±9 pg/islet/min) or the combination of 20 mM glucose plus 1 mM carbachol (358±85 pg/islet/min) were also significantly decreased when compared with fresh islets. We conclude from these studies that the process of culturing islets for one day in CMRL-1066 significantly decreases islet cell PI hydrolysis and insulin secretory responsiveness. These observations may help to explain the discordant conclusions reached concerning the involvement of PI hydrolysis and protein kinase C activation in the regulation of insulin release from freshly isolated versus cultured islets.

Cadmium disrupts the signal transduction pathway of both inhibitory and stimulatory receptors regulating chloride secretion in the shark rectal gland

The heavy metal cadmium causes nephrotoxicity and alters the transport function of epithelial cells. In the shark rectal gland, chloride secretion is regulated by secretagogues and inhibitors acting through receptors coupled to G proteins and the cyclic AMP-protein kinase A pathway. We examined the effects of cadmium on the response to the inhibitory peptide somatostatin (SRIF), and to the stimulatory secretagogues forskolin and vasoactive intestinal peptide (VIP). In control experiments, SRIF (100 nM) entirely inhibited the chloride secretory response to 10 microM forskolin (maximum chloride secretion with forskolin 1984 +/- 176 microEq/h/g; with forskolin + SRIF 466 +/- 93 microEq/h/g, P < 0.001). Cadmium (25 microM) entirely reversed the inhibitory response to SRIF (chloride secretion 2143 +/- 222 microEq/h/g) and caused an overshoot (2917 +/- 293 microEq/h/g) that exceeded the response to forskolin (P < 0.01). Cadmium also enhanced forskolin-stimulated chloride secretion (2628 +/- 418 vs. 1673 +/- 340 microEq/h/g, P < 0.02) and reversed the declining phase of the forskolin response. Cadmium had a concentration-dependent, biphasic effect on the response to VIP. Cd (10-100 microM) increased both chloride secretion and tissue cyclic AMP content, whereas higher concentrations (1 mM) inhibited chloride secretion and cyclic AMP accumulation. Our findings provide evidence that Cd disrupts the signal transduction pathways of both inhibitory receptors and secretagogues regulating cAMP mediated transport in an intact epithelia. The results are consistent with direct effects of cadmium on adenylate cyclase and/or phosphodiesterase activity in this marine epithelial model.