Bis(benzylisoquinoline) analogs of tetrandrine block L-type calcium channels: evidence for interaction at the diltiazem-binding site.

Bis(benzylisoquinoline) alkaloids block Ca2+ uptake through the L-type Ca2+ channel and modulate binding of ligands to four distinct sites (dihydropyridine, benzothiazepine, aralkylamine, and (diphenylbutyl)piperidine) in the Ca2+ entry blocker receptor complex of the channel. These alkaloids are structural analogs of tetrandrine, which has previously been demonstrated to block the L-type Ca2+ channel through interaction at the benzothiazepine (diltiazem) site (King et al., 1988). Different alkaloid conformational classes display either alpha-beta, beta-alpha, alpha-alpha, or beta-beta stereochemistry at the two chiral isoquinoline carbons. Compounds from all four classes were tested for their ability to interact with Ca2+ entry blocker ligands. All analogs completely inhibit diltiazem binding, but many only partially inhibit D-600 and fluspirilene binding. For dihydropyridine binding, the compounds show either stimulation or inhibition or exhibit no effect. This profile is quite different from the interaction displayed by diltiazem or tetrandrine. Scatchard analyses show effects predominantly on Kd for diltiazem, D-600, and PN200-110 binding. Representative conformers do not effect diltiazem dissociation rates but alter dissociation kinetics of ligands which bind to the other three sites. A correlation of the ability of these compounds to inhibit Ca2+ uptake through the L-type Ca2+ channel in GH3 cells exists only with their inhibition of diltiazem binding but not with inhibition of binding of ligands representing other classes of Ca2+ entry blockers. These data, taken together, indicate that a variety of bis(benzylisoquinoline) congeners act to block the L-type Ca2+ channel by binding to the benzothiazepine site on the channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride analogs inhibit L-type calcium channels and display calcium entry blocker activity.

Three structural classes of commonly used amiloride analogs, molecules derivatized at the terminal guanidino-nitrogen, the five-position pyrazinoyl-nitrogen, or di-substituted at both of these positions, inhibit binding of the L-type Ca2+ channel modulators diltiazem, gallopamil, and nitrendipine to porcine cardiac sarcolemmal membrane vesicles. The rank order of inhibitory potencies among the various derivatives tested is well defined with amiloride being the least potent. Saturation binding studies indicate that inhibition of ligand binding results primarily from effects on Kd. Ligand dissociation measurements suggest that amiloride derivatives do not associate directly at any of the known sites in the Ca2+ entry blocker receptor complex. In addition, these compounds do not compete at the "Ca2+ coordination site" within the channel. However, studies with inorganic and substituted diphenylbutylpiperidine Ca2+ entry blockers reveal that amiloride analogs interact at a site on the channel where metal ions bind and occlude the pore. Photolysis experiments performed with amiloride photoaffinity reagents confirm that a specific interaction occurs between such probes and the channel protein. Upon photolysis, these agents produce concentration- and time-dependent irreversible inactivation of Ca2+ entry blocker binding activities, which can be protected against by either verapamil or diltiazem. 45Ca2+ flux and voltage-clamp experiments performed with GH3 anterior pituitary cells demonstrate that amiloride-like compounds inhibit L-type Ca2+ channels directly. Moreover, these compounds block contraction of isolated vascular tissue in pharmacological assays. Electrophysiological experiments indicate that they also inhibit T-type Ca2+ channels in GH3 cells. Taken together, these results demonstrate unequivocally that amiloride analogs display significant Ca2+ entry blocker activity in both ligand binding and functional assays. This property, therefore, can seriously complicate the interpretation of many in vitro and in vivo studies where amiloride analogs are used to elicit inhibition of other transport systems (e.g. Na-Ca and Na-H exchange).

High levels of sodium-calcium exchange in vascular smooth muscle sarcolemmal membrane vesicles.

Membrane vesicles which exhibit high levels of Nai-dependent Ca2+ uptake have been prepared from either porcine or bovine aortic smooth muscle. These membranes are identified as being of sarcolemmal origin by enrichment of marker activities associated with the sarcolemma (e.g., binding of the ligands PN 200-110, iodocyanopindolol, and ouabain). The Vmax of Na-Ca exchange in the two aortic sarcolemmal preparations [0.5-3.5 nmol s-1 (mg of protein)-1] is significantly higher than that previously reported with membrane preparations derived from visceral and vascular smooth muscle and compares favorably with maximal values recorded in cardiac sarcolemmal membrane vesicles [5-20 nmol-1 s-1 (mg of protein)-1] under identical experimental conditions. The Km of Ca2+ (15 +/- 5 microM) and the Km of Na+ (15 +/- 7 mM) are similar values as determined in heart. Aortic and cardiac Na-Ca exchange activities are equivalent in their sensitivity to inhibition by La3+ and two known classes of mechanism-based organic blockers of transport activity (i.e., amiloride analogues and bepridil-like agents). Both also display electrogenic behavior. However, Li+, K+, and choline all inhibit the smooth muscle transporter with markedly greater potency than found in heart, and intravesicular Ca2+ does not affect transport activity in smooth muscle membranes as it does in the cardiac system. When maximal transport velocities are compared, aortic membrane vesicles have 3-6-fold higher Na-Ca exchange than sarcolemmal Ca2+-ATPase Ca2+ transporting capacities.(ABSTRACT TRUNCATED AT 250 WORDS)

Substituted diphenylbutylpiperidines bind to a unique high affinity site on the L-type calcium channel. Evidence for a fourth site in the cardiac calcium entry blocker receptor complex.

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.

Regional distribution of pancreatic polypeptide cells in the 21-day fetal rat pancreas.

21-day fetal rat pancreata were stained with the unlabeled antibody peroxidase-antiperoxidase technique using bovine pancreatic polypeptide as the primary antibody. Total counts of pancreatic polypeptide cells were made over the entire pancreas. It was found that the head region contained the greatest number of pancreatic polypeptide cells with the body next and the tail having the smallest number. The pancreatic polypeptide cells of the body were concentrated in the portion closest to the distal duodenum. This distribution pattern seems to support the suggested role of pancreatic polypeptide on the physiological function of the digestive tract.

Further evidence for an argyrophilic fourth cell type in the pancreatic islet of the rat.

An argyrophilic fourth cell type in fetal and adult rat pancreatic islets can be identifed by using a modification of the Grimelius silver statin. This cell is much more abundant in the fetal pancreas than in the adult. By employing the modified silver technique followed by restaining with the indirect immunofluorescent procedure for somatostatin, the content of this argyrophilic fourth cell was studied further. Comparison of these histochemical studies demonstrated that somatostatin was not located in the fourth cell of either the adult or fetal rat pancreas. These results indicate that the D-cell and the fourth cell type are not the same cell. Thus far the only product associated with this argyrophilic cell is pancreatic polypeptide. As a result this cell probably represents the PP-cell of the Wiesbaden classification.

The staining of pancreatic delta cells for light microscopy.

Three staining techniques--Hellerström-Hellman alcoholic silver nitrate, pseudoisocyanin, and the immunofluorescent technique for somatostatin--were compared to determine whether they are staining the same cell in the pancreatic islet of the rat. These comparisons were made by using staining and restaining procedures on the same islet. The results demonstrate that all three procedures stain the delta cell of the rat pancreatic islet. Some advantages of using pseudoisocyanin are discussed.