Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells.

We have examined the interaction between muscarinic and alpha(2)-adrenergic receptor activation on inositol phosphate (IP) formation in the nonpigmented cells of the ciliary body epithelium (NPE cells) of the rabbit. We have compared these changes with those previously observed in the intracellular free Ca(2+) concentration. Whereas muscarinic receptor activation causes an increase in intracellular Ca(2+) and IP formation, activation of alpha(2)-receptors does not significantly increase either intracellular Ca(2+) or IPs over basal levels. However, simultaneous activation of muscarinic and alpha(2)-adrenergic receptors with the specific agonists carbachol and UK-14304 produces massive Ca(2+) increases and results in a synergistic increase in IP formation. This synergistic IP formation is inhibited by both muscarinic and alpha(2)-adrenergic receptor antagonists as well as by pertussis toxin and an inhibitor of phospholipase C. IP formation is predominantly independent of intracellular Ca(2+), because it is decreased but not prevented by blocking the entry of Ca(2+) with LaCl(3) or chelating intracellular Ca(2+) with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca(2+) resulting from simultaneous activation of muscarinic and alpha(2)-adrenergic receptors.

Contractile role of M2 and M3 muscarinic receptors in gastrointestinal smooth muscle.

Muscarinic agonists elicit contraction through M3 receptors in most isolated preparations of gastrointestinal smooth muscle, and not surprisingly, several investigators have identified M3 receptors in smooth muscle using biochemical, immunological and molecular biological methods. However, these studies have also shown that the M2 receptor outnumbers the M3 by a factor of about four in most instances. In smooth muscle, M3 receptors mediate phosphoinositide hydrolysis and Ca2+ mobilization, whereas M2 receptors mediate an inhibition of cAMP accumulation. The inhibitory effect of the M2 receptor on cAMP levels suggests an indirect role for this receptor; namely, an inhibition of the relaxant action of cAMP-stimulating agents. Such a function has been rigorously demonstrated in an experimental paradigm where gastrointestinal smooth muscle is first incubated with 4-DAMP mustard to inactivate M3 receptors during a Treatment Phase, and subsequently, the contractile activity of muscarinic agonists is characterized during a Test Phase in the presence of histamine and a relaxant agent. When present together, histamine and the relaxant agent (e.g., isoproterenol or forskolin) have no net contractile effect because their actions oppose one another. However, under these conditions, muscarinic agonists elicit a highly potent contractile response through the M2 receptor, presumably by inhibiting the relaxant action of isoproterenol or forskolin on histamine-induced contractions. This contractile response is pertussis toxin-sensitive, unlike the standard contractile response to muscarinic agonists, which is pertussis toxin-insensitive. When measured under standard conditions (i.e., in the absence of histamine and without 4-DAMP mustard-treatment), the contractile response to muscarinic agonists is moderately sensitive to pertussis toxin if isoproterenol or forskolin is present. Also, pertussis toxin-treatment enhances the relaxant action of isoproterenol in the field-stimulated guinea pig ileum. These results demonstrate that endogenous acetylcholine can activate M2 receptors to inhibit the relaxant effects of beta-adrenoceptor activation on M3 receptor-mediated contractions. An operational model for the interaction between M2 and M3 receptors shows that competitive antagonism of the interactive response resembles an M3 profile under most conditions, making it difficult to detect the contribution of the M2 receptor.

Processing and ligand-induced modifications of the V2 vasopressin receptor.

Synthesis, processing and agonist-induced modifications of the V2 vasopressin receptor were examined in stably or transiently transfected HEK293 cells. Metabolic labeling with S methionine for 30 min revealed a predominant precursor protein which subsequently gave rise to the mature receptor on the cell surface. Maturation of the receptor was unrelated to glycosylation suggesting that it was the consequence of protein refolding. In addition to monomeric forms of V2 receptor protein, oligomers of the precursor protein were also detected in SDS-PAGE. These oligomers seemed to be dimers and tetrameres, and were more apparent in transiently transfected cells that produced higher quantities of protein then stably transfected cells. No oligomers of the mature receptor were detected, and co-transfection of the wild type with a mutant V2 receptor lacking G-protein coupling activity did not alter the function of the wild type receptor. These results indicated that the formation of oligomeric was most likely a consequence of overproduction of the protein and not a required step for receptor function. Addition of vasopressin promoted phosphorylation and sequestration of the wild type receptor, and of the R137H mutant receptor which lacks coupling to G proteins. Activation of protein kinases A or C did not result in phosphorylation of un-occupied receptor. Phosphate incorporated into the protein was stable in the continuous presence of the ligand despite sequestration of the receptor protein. Deletion of the last 14 amino acids abolished receptor phosphorylation but not sequestration and desensitization, indicating that these two processes are not dependent on protein phosphorylation. Additionally, phosphorylation and sequestration of the R137H mutant receptor revealed that phosphorylation and sequestration does not require coupling to Gs. The wild type V2 vasopressin receptor was found to be palmitoylated at two cysteines at the carboxyl terminus. Either cysteine could be palmitoylated independently of each other and the presence of at least one was required to obtain receptor expression similar to the wild type. The turnover of the palmitic acid incorporated into the receptor was not altered by the addition of vasopressin demonstrating that this post-translational modification of the receptor was not altered by the ligand-promoted phosphorylation of the protein.

Enhanced increase of plasma sodium pump inhibitory activity to saline expansion in vagosympathectomized and anaesthetized dogs.

To investigate whether plasma sodium pump inhibitory activity is controlled by cardiopulmonary and aortic baroreceptors, mean arterial pressure, right atrial pressure, sodium and water balances, plasma renin activity, plasma aldosterone concentration and plasma antinatriferic activity (PAA; plasma sodium pump inhibitory activity) were determined before, during and after Ringer volume expansion (10% of body wt) in anaesthetized dogs. Animals were studied with intact reflexes (CTR, n = 7) and after acute cervical bilateral vagosympathetic denervation (VGT, n = 8). With the exception of PAA, none of the parameters were different between groups before, during or after Ringer volume expansion. The PAA (microA cm-2) was similar for both groups before expansion and before either sham (CTR) or vagosympathectomy (VGT) was performed (CTR = 3.6 +/- 0.4 vs. VGT = 4.3 +/- 0.3). Compared to baseline, PAA at the end of the volume expansion phase increased in both groups (CTR = 6.1 +/- 0.8, P < 0.05; VGT = 9.1 +/- 0.7, P < 0.0005); however, this PAA value was significantly greater in the VGT group than in the CTR group (P < 0.01). At the end of the post-expansion phase, PAA levels returned toward baseline in both groups (CTR = 4.4 +/- 0.5 vs. VGT = 4.8 +/- 0.2; n.s. vs. baseline); however, this PAA value in the CTR group was not significantly different from its pak value. The present data confirm that PAA is increased in response to saline volume expansion, and suggest that PAA synthesis and/or release is under inhibitory vagosympathetic control during saline volume expansion.

Stimulation of cyclic AMP accumulation and phosphoinositide hydrolysis by M3 muscarinic receptors in the rat peripheral lung.

The effects of oxotremorine-M (oxo-M), a muscarinic agonist, on cyclic AMP (cAMP) accumulation in slices of the rat peripheral lung were investigated. Oxo-M stimulated cAMP accumulation in a concentration-dependent manner with an EC50 value of 4.2 microM and a maximal effect of 2.4 +/- 0.39-fold over basal. In the presence of forskolin (25 microM), the maximal effect of oxo-M was increased to 14.1 +/- 4.0-fold over basal. Forskolin alone caused a 5.9 +/- 2.2-fold increase in cAMP relative to basal; therefore, the combination of both drugs was more than additive. The effects of oxo-M on cAMP accumulation were unaffected by tetrodotoxin, indicating that the action of oxo-M was not mediated by neuronal release of neurotransmitters. Oxo-M had a small inhibitory effect on cAMP in a homogenate preparation, indicating that the stimulatory response to oxo-M in slices of the lung is not due to direct stimulation of adenylyl cyclase. Characterization of the oxo-M potentiation of forskolin-stimulated cAMP accumulation using different muscarinic antagonists yielded calculated pKB values that agreed with binding affinities for the M3 subtype. Oxo-M elicited phosphoinositide hydrolysis in the lung, and the nature of the antagonism of this response was also consistent with that expected for an M3-mediated response. cAMP accumulation in the presence of oxo-M (100 microM), forskolin (12 microM), or both drugs combined was inhibited by indomethacin (1 microM). These results demonstrate that the M3 receptor stimulates cAMP accumulation and phosphoinositide hydrolysis in the rat peripheral lung, and the mechanism for cAMP stimulation may involve arachidonic acid metabolites.