Background & Aims: Heterotrimeric G proteins are important in growth-regulating signal transduction. The aim of this study was to characterize the relative expression of G protein a subunits in rat colonocytes, colonocyte antipodal plasma membranes, and colonic neoplasms. Methods: Antipodal plasma membranes were prepared from isolated colonocytes, Azoxymethane was administered to rats to induce colonic neoplasms. K-ras mutations in the neoplasms were determined by oligonucleotide hybridization and confirmed by primer mediated-restriction fragment length polymorphism. Colonocyte and tumor homogenates or membranes were probed for G alpha subunits by Western blotting with isoform-specific antibodies. Results: The expressions of G alpha i2, alpha i3, and alpha q/11 were significantly enriched in the basolateral compared with brush border fraction of colonic antipodal plasma membranes. In neoplasms without K-ras mutations, the expression of G alpha i2 increased 4-fold, G alpha s(long) increased 2.5-fold, and G alpha i3 increased 1.5-2-fold. Expression did not differ among tumor grades. K-ras mutations were associated with lowered expression of G proteins, especially G alpha o. Conclusions: In colonocytes, G alpha subunits are localized primarily in basolateral plasma membranes. The increased expressions of G alpha i2 and, to a lesser degree, G alpha i3 and G alpha s(long) in tumors was independent of tumor grade but was modulated by the presence of K-ras mutations.

The present studies were conducted to determine whether [3H]quinuclidinyl benzilate binding in rat colonic membranes and/or carbachol-mediated stimulation of particulate guanylate cyclase were altered by changes in vitamin D status. EC50 values for the stimulation of colonic guanylate cyclase by carbachol were found to be significantly greater in vitamin D-deficient rats compared to their D-sufficient counterparts. Concomitantly, the density of receptors (Bmax) were significantly lower, and dissociation constants (Kd) were significantly higher in D-deficient colonic membranes. In vitamin D-repleted animals, moreover, all of these aforementioned alterations were at least partially corrected.

Recent studies have implicated protein kinase-C (PKC) in the regulation of guanylate cyclase in several cell types. In view of prior experiments by our laboratory which have demonstrated that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] can activate PKC in CaCo-2 cells, it was of interest to determine whether this secosteroid influenced particulate guanylate cyclase and, if so, to determine which isoforms of PKC were involved. To address these issues, CaCo-2 cells were treated with 1 alpha,25-(OH)2D3 or other agents (see below), and crude membranes prepared from these cells were assayed for guanylate cyclase activity. In several experiments, agents were added directly to isolated membranes, and guanylate cyclase activity was then assayed. These studies demonstrated that 1) the addition of 1 alpha,25-(OH)2D3 or 12-O-tetradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, to intact CaCo-2 cells stimulated particulate guanylate cyclase activity in a time- and concentration-dependent manner; 2) these agents induced the translocation of PKC alpha, but not PKC zeta, from the cytosolic to the membrane fraction of these cells; 3) preincubation of cells with staurosporine (50 nM), a PKC inhibitor, or U73122 (10 microM), an inhibitor of phospholipase-C-dependent processes, significantly reduced (P < 0.05) the stimulatory effect of 1 alpha,25-(OH)2D3 (3 nM) on guanylate cyclase; 4) preincubation of isolated membranes with TPA, calcium, and Mg(2+)-ATP increased guanylate cyclase activity, an affect that was augmented by purified rat brain PKC and inhibited by the PKC inhibitor peptide, PKC-(19-36); and 5) selective down-regulation of PKC alpha by treatment of cells with TPA (200 nM) for 24 h concomitantly abolished the activation of guanylate cyclase by 1 alpha,25-(OH)2D3. Taken together, these studies demonstrate that 1 alpha,25-(OH)2D3 activates particulate guanylate cyclase at least in part via a PKC alpha-dependent mechanism.

The phosphoinositide signal transduction pathway mediates important processes in intestinal physiology, yet the key enzyme, phosphoinositide-specific phospholipase C (PI-PLC), is not well-characterized in the colon. PI-PLC activity was examined in rat colonic membranes using exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate, and beta-glycerophosphate to suppress degradation of substrate or product. The activity of membrane PI-PLC increased 6-fold with the addition of alamethicin, and a further 2-3-fold enhancement was observed with 10 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 microM adenosine 5'-[gamma-thio]-triphosphate failed to stimulate PI-PLC activity, and guanosine 5'-[beta-thio]diphosphate inhibited activity. The response of membrane PI-PLC to Ca2+ was biphasic, while > 0.5 mM Mg2+ was inhibitory with or without GTP[S]. Comparable total PI-PLC activities and responses to GTP[S] and Ca2+ were observed in purified brush-border and basolateral membranes. Western immunoblots probed with monoclonal antibodies to PLC isoenzymes PLC-beta 1, -gamma 1 and -delta 1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-delta 1 isoform, with small amounts of PLC-gamma 1 present but no detectable PLC-beta 1. PLC-gamma 1 was the major isoform detected in cytosol.

P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues. P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport [3H]daunomycin. In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of [3H]daunomycin and [3H]vinblastine. Accumulation of both cytotoxic agents was stimulated by ATP, exhibited temperature dependence and osmotic sensitivity, and followed Michaelis-Menten kinetics. Alterations in CMV lipid fluidity were induced by the known fluidizers, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) and benzyl alcohol, and were assessed by fluorescence polarization techniques using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). Both A2C (2.5-5.0 microM) and benzyl alcohol (10-20 mM) produced a dose-dependent increase in CMV lipid fluidity. Moreover, both fluidizers, at the above doses, significantly inhibited (p < 0.05) the ATP-dependent accumulation of [3H]daunomycin. [3H]Vinblastine accumulation was also inhibited by A2C (p < 0.05). Lower doses of A2C (0.6 microM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation. Kinetic analysis revealed that A2C (5.0 microM) noncompetitively inhibited [3H]daunomycin accumulation and uncompetitively inhibited [3H]vinblastine accumulation with apparent Ki values of approximately 1.5 and approximately 1.2 microM, respectively. Verapamil competitively inhibited P-gp-mediated accumulation of [3H]daunomycin but failed to alter the fluidity of CMV. Taken together, the present results demonstrate that while increases in membrane fluidity of CMV are not necessarily required to inhibit P-gp-mediated drug accumulation, they can inhibit these processes, at least in CMV. Alterations in the physical state of CMV, therefore, appear to be at least one important modulator of P-gp function.

Experiments were conducted, using a nonspecific lipid transfer protein, to vary the cholesterol/phospholipid molar ratio of rat proximal small intestinal microvillus membranes in order to assess the possible role of cholesterol in modulating enzymatic activities of this plasma membrane. Cholesterol/phospholipid molar ratios from 0.71 to 1.30 were produced from a normal value of 1.05 by incubation with the transfer protein and an excess of either phosphatidylcholine or cholesterol/phosphatidylcholine liposomes for 60 min at 37 degrees C. Cholesterol loading or depletion of the membranes was accompanied by a decrease or increase, respectively, in their lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the lipid-soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Increasing the cholesterol/phospholipid molar ratio also decreased alkaline phosphatase specific activity by approximately 20-30%, whereas decreasing this ratio increased this enzymatic activity by 20-30%. Sucrase, maltase, and lactase specific activities were not affected in these same preparations. Since the changes in alkaline phosphatase activity could be secondary to alterations in fluidity, cholesterol, or both, additional experiments were performed using benzyl alcohol, a known fluidizer. Benzyl alcohol (25 mM) restored the fluidity of cholesterol-enriched preparations to control levels, did not change the cholesterol/phospholipid molar ratio, and failed to alter alkaline phosphatase activity. These findings, therefore, indicate that alterations in the cholesterol content and cholesterol/phospholipid molar ratio of microvillus membranes can modulate alkaline phosphatase but not sucrase, maltase, or lactase activities. Moreover, membrane fluidity does not appear to be an important physiological regulator of these enzymatic activities.

In dispersed acini from rat pancreas, binding of 125I-labeled vasoactive intestinal peptide and 125I-labeled secretin was relatively rapid, reversible, saturable, and temperature dependent. The rate of dissociation of bound 125I-labeled peptide was not a function of the concentration of free vasoactive intestinal peptide or secretin, indicating that the apparent affinities of these labeled peptides for their binding sites do not depend on the extent of receptor occupation. Four classes of receptors are required to account for the actions of vasoactive intestinal peptide and secretin on enzyme secretion, cellular cAMP, and binding of 125I-vasoactive intestinal peptide and 125I-secretin. One class has a high affinity for vasoactive intestinal peptide, and occupation of this class of receptors causes increased cellular cAMP and stimulation of amylase secretion. A second class has a low affinity for vasoactive intestinal peptide and for secretin, and occupation of these receptors does not cause changes in cAMP or amylase secretion. A third class of receptors has a high affinity for secretin, and occupation of these receptors causes increased cAMP and stimulation of amylase secretion. A fourth class of receptors has a low affinity for secretin, and occupation of these receptors causes stimulation of amylase secretion by a non-cAMP-mediated mechanism.

Cimetidine has been shown to be an effective drug for healing peptic ulcers and for preventing their recurrence.1 In most clinical trials cimetidine has been associated with few side effects of clinical importance. Early toxicologic studies with cimetidine, however, showed a decrease in the weight of the prostate and seminal vesicles in rats and dogs,2 , 3 suggesting the presence of antiandrogen activity. Later studies demonstrated that cimetidine could inhibit the binding of dihydrotestosterone to androgen receptors in the prostates of rats and the kidneys of mice.3 4 5 6 Subsequently, the occurrence of gynecomastia,7 8 9 10 especially in pathologic hypersecretory states,7 , 11 , 12 and occasional cases of impotence . . .