Mary Ann Kowatch

Effect of specific membrane perturbations on alpha1-adrenergic and muscarinic-cholinergic signal transduction in rat parotid cell aggregates

Alpha 1-adrenergic and muscarinic-cholinergic stimulated IP3 production and calcium mobilization are inhibited by treatment of parotid cell aggregates with methanol, hydrogen peroxide and saponin. Only methanol exerts an effect on binding to receptors. In most cases a close correspondence exists between inhibition of alpha 1-adrenergic and muscarini-cholinergic responses as well as inhibition of IP3 production and calcium mobilization. G-protein dependent signal transduction, therefore appears to be quite sensitive to plasma membrane perturbation and membrane active agents may provide useful tools for elucidation of transduction mechanisms and their regulation.

Similar effects of saponin treatment and aging on coupling of α1-adrenergic receptor-G-protein

Abstract The effects of the nonionic detergent saponin on α1-adrenergic signal transduction were investigated using rat parotid cells and membrane preparations. Fifty μM epinephrine-stimulated 45Ca2+ efflux and inositol 1,4,5-triphosphate (Ins[1,4,5]P3) production in adult parotid cells were significantly decreased after saponin treatment. Saponin did not alter the concentration of α1-adrenergic receptors labeled by [3H]prazosin, but significantly reduced the guanosine imido diphosphate (GppNHp)-induced shift from high to low affinity sites. Fifty μM epinephrine-stimulated high affinity GTPase activity was also reduced by saponin treatment. These data suggest that reduced α1-adrenergic receptor-stimulated functional responsiveness following saponin treatment may be due to impaired uncoupling of receptor-G-protein complexes.

Evidence for an alteration in the microsomal Ca2+ release mechanism in senescent rat parotid acinar cells

Previously, we have shown that Ca2+ mobilization following an alpha 1-adrenergic receptor stimulus is reduced in parotid acinar cells from senescent rats as a result of an altered ability of inositol 1,4,5-trisphosphate (IP3) to induce Ca2+ release from a non-mitochondrial, intracellular Ca2+ store (Ishikawa, Y., et al. Biochim. Biophys. Acta 968, 203-210). We have used this model to examine the IP3-induced Ca2+ release mechanism in these cells. 45Ca2+ efflux, after exposure to (-) epinephrine, from cells of young adult (3-6 months) rats was approx. 2-fold that observed from cells from older animals (approx. 24 months) either in the presence or absence of extracellular Ca2+. Similarly, cytosolic Ca2+ levels were greater in cells of young adult rats under these same incubation conditions. However, microsomal membrane preparations, from both age groups displayed similar IP3 binding sites (Kd approximately 90 nM, Bmax approximately 850 fmol/mg protein) and ATP-dependent Ca2+ transport ability (approx. 8 nmol/mg protein.min -1). These data suggest that there is an alteration in the IP3-induced Ca2+ release mechanism in microsomal membranes of parotid glands from senescent rats which may account for the decreased Ca2+ release seen after agonist stimulation of this tissue.

Alpha1-adrenergic and muscarinic-cholinergic stimulated inositol trisphosphate production may proceed through different post-receptor signal transduction pathways in parotid acini

Maximal stimulation of inositol 1,4,5 trisphosphate (IP3) production by epinephrine and carbachol in rat parotid cell aggregates is additive when the two agents are employed simultaneously. The additive response proceeds through both the alpha 1-adrenergic and muscarinic-cholinergic signal transduction pathways. It is critical that IP3 be measured by a radioreceptor assay, since when cells are labeled with 3H-inositol and IP3 determined by ion exchange chromatography, additivity is not detectable. Reasons for the discrepancy between methods are discussed. These results, coupled with the differential sensitivity of the alpha 1-adrenergic and muscarinic cholinergic pathways to neomycin and aging, suggest that they may be dissociated at the post-receptor level.

α1-Adrenoceptors in parotid cells: age does not alter the ratio of α1A and α1B subtypes

Abstract Epinephrine stimulation of 45Ca2+ efflux and inositol 1,4,5-trisphosphate ((1,4,5)IP3) production in parotid cell aggregates from mature rats was greatly inhibited (≈70%) by WB 4101 and 5-methylurapidil as compared to a small decrease by chloroethylclonidine (≈30%). The combination of WB 4101 or 5-methylurapidil and chlorophylclonidine completely blocked the action of epinephrine. The same relative inhibition was observed with senescent animals. The results suggest (1) that rat parotids contain both α1-adrenoceptor subtypes, i.e., α1A and α1B, in an approximate functional ratio of 70:30, (2) that this relative ratio is not altered during aging, and (3) that both receptors partially mediate 45Ca2+ efflux and (1,4,5)IP3 production in this system.

Partial restoration of impaired alpha1-adrenergic responsiveness in parotid cells of aged rats by S-adenosylmethionine treatment

The age related decrease in alpha1-adrenergic stimulated inositol 1,4,5 trisphosphate (IP3) production in parotid cells of aged rats can be partially restored by treatment with S-adenosylmethionine (SAM). This effect is completely blocked by S-adenosyl homocysteine (SAH) and occurs in association with an increase in the conversion of phosphatidylethanolamine to phosphatidylcholine and a decrease in membrane viscosity. In contrast, SAM treatment actually inhibits stimulated IP3 production in cells of young rats. The membrane viscosity of these cells is lower than that of those from aged rats. Although conversion of phosphatidylethanolamine to phosphatidylcholine is enhanced, no further decrease in membrane viscosity is elicited in young cell preparations. These findings suggest that age changes in the membrane environment may result in impaired alpha1-adrenergic signal transduction and that such alterations may be at least partially reversible by SAM treatment.

Behavior of angiotensin octapeptides and dinitrophenyl-angiotensin during chromatography on polyacrylamide and dextran gels

The behavior of the synthetic angiotensin II octapeptides (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, molecular weight 1046, and its Asn1-Val5 congener, molecular weight 1031) have been studied during gel chromatography on cross-linked polyacrylamide (Bio-Gel, P-2), and dextran (Sephadex G-15). In the case of the asparagine peptide adsorption was observed with both media. On polyacrylamide adsorption was marked at acid pH (0.05 N HCl), but at pH 5.5 in 0.1 M pyridinium acetate the asparagine compound could be separated from and emerged between a larger peptide (bacitracin, molecular weight 1411) and smaller molecules (pyridine—Cu2+ complex; Na+). On dextran gel asparagine1-angiotensin was retarded due to adsorption not only at acid pH but also in 0.1 M pyridinium acetate, with adsorption even more marked in dilute alkali (0.01 N NH4OH). Under none of these conditions was it possible with ordinary dextran gel to separate asparagine1-angiotensin from amino acids or salts. In sharp contrast, the aspartic acid1-angiotensin was excluded from G-15 at alkaline pH, and the two angiotensins were thus readily separated from one another under these conditions. Adsorption of the asparagine1-angiotensin was nearly abolished by 1 M pyridine. The separation of the two angiotensins under these conditions is attributed to the greater negative charge of the aspartic acid1-argiotensin which results in exclusion from the gel and overcomes the “aromatic” adsorption of the remainder of the molecule. The dinitrophenyl (DNP) derivative of asparagine1-angiotensin is readily separable from DNP-amino acids by chromatography in absolute methanol through an alkylated dextran gel (Sephadex LH-20).

Double isotope assay of nanogram quantities of peptides as their dinitrophenyl derivatives: Angiotensin and related compounds

Abstract A double-isotope derivative assay has been developed which allows quantitation of angiotensin II on a nanogram scale. The peptide is reacted with 3H-labeled 1-fluoro-2,4 dinitrobenzene (3H-FDNB) to yield a trisubstituted dinitrophenyl (DNP) derivative under conditions in which it has been shown that reaction of the peptide with FDNB is quantitative. Separately prepared 14C-DNP-angiotensin is added as indicator and nonlabeled DNP peptide as carrier. Excess reagent is removed by extraction with toluene, and the peptide is extracted into ethyl acetate and subjected to TLC on silica gel. The yellow DNP derivative is eluted, converted to its methyl ester with methanol-HCl, rechromatographed, eluted, and its 3H and 14C radioactivity determined in a liquid scintillation spectrometer. The reagent blank is zero. Yields of derivative are quantitative. Recoveries are variable (20–40%) but accurate correction is provided by use of the 14C indicator. With 3H-FDNB of specific activity of 200 μC/μ m two or more nanograms can be accurately determined. Angiotensin II has also been assayed on a nanogram scale after carboxypeptidase degradation to its des-phenylalanine heptapeptide, using appropriate heptapeptide 14C indicator and carrier. The DNP derivatives of several peptides related to angiotensin I and II have been prepared and shown to be separable by TLC. The 3H-DNP derivative method should be readily adaptable to the measurement of a variety of other peptides, including angiotensin I, and may provide the basis for an assay of renin in plasma.