An HIV-1 protease screening assay using a non-infectious proviral clone.

An in-vitro assay was developed to screen for HIV-1 protease inhibitors using a non-infectious proviral clone (X19) with a deletion in the envelope gene (Ratner et al., 1991). The proviral clone, X19, was expressed transiently in the COS 7 cell line. The virus was able to replicate as assessed by the presence of p24 in the supernatants, yet the virions produced did not infect CD4 positive cells. To determine the effect of a known protease inhibitor on p24 antigen production, PD 148310 (a Ro 31-8959 analog) was added immediately after transfection of the COS 7 cells. Virus particles were produced maximally after 24 h and cell supernatants were assayed for p24 antigen production using a p24 ELISA assay. PD 148310 inhibited p24 release in a dose-dependent manner with an IC50 of 23.6 nM. Western blot analysis of the supernatants using a mouse monoclonal antibody against p24 confirmed the presence of a well-defined p24 band in the control lane. At 1000 nM of PD 148310 the p24 band was not detectable, leaving only the unprocessed p55 Gag precursor. This technique is a useful tool to screen for potential HIV-1 protease inhibitors.

Inhibition of guinea-pig oxyntic cell function by adenosine and prostaglandins.

Adenosine and prostaglandins (PGs) are known inhibitors of oxyntic cell function. Using quantitative cytochemistry of hydroxyl ion production (HIP) in guinea-pig oxyntic cells, we examined the effects of adenosine and PGs on secretagogue-stimulated HIP. Adenosine (10(-6) M) inhibited the actions of histamine (10(-14) M) and gastrin (2.5 X 10(-12) M) by 69 and 67%, respectively, but not that of dibutyryl cyclic AMP (10(-16) M) or carbachol (10(-9) M). These observations suggest that adenosine does not influence the Ca++-dependent pathway of carbachol action and that the adenosine activity precedes the generation of cyclic AMP. Adenosine and related analogs, N6-L-phenylisopropyladenosine and 5-N-ethylcarboxam-idoadenosine (10(-12) to 10(-14) M), inhibited histamine-stimulated HIP (10(-14) M) in the following order: N6-L-phenylisopropyladenosine greater than 5-N-ethylcarboxamidoadenosine greater than adenosin. The adenosine antagonist, 1,3-diethylphenylxanthine (10(-6) M), reversed the inhibitory effects of adenosine. Exogenous PGE2 (10(-6) M) also inhibited histamine- and gastrin-stimulated HIP by 65 and 55%, respectively. Indomethacin (10(-6) M) and flurbiprofen (10(-6) M), PG synthesis inhibitors, potentiated the action of histamine by 175 and 159%, respectively. Adenosine was incapable of reversing this potentiated effect. These data indicate that adenosine and related analogs are inhibitors of oxyntic cell HIP and suggest that these biological properties are mediated by binding to a cell surface receptor and thereby regulating oxyntic cell adenylate cyclase activity. The more potent properties of N6-L-phenylisopropyladenosine as compared to 5-N-ethylcarboxamidoadenosine are consistent with activity at the high-affinity surface adenosine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of gastrin in guinea pig oxyntic cells. Studies using quantitative cytochemistry.

The mechanism of action of gastrin was investigated using cytochemical quantitation of hydroxyl ion production (HIP) in guinea pig gastric oxyntic mucosa. The reaction depends upon the trapping of OH ions produced during gastric stimulation and is blocked by the benzimidazole, Hassle 149/94, which inhibits the K+ + H+-ATPase and by acetazolamide, an inhibitor of carbonic anhydrase activity. It is thus a measure of hydroxyl ions produced during stimulation of the oxyntic cell and reflects upon hydrogen ion production. Gastrin (2.5 X 10(-16) -2.5 X 10(-12) M) caused a linear dose-dependent stimulation of HIP in the oxyntic cells. The response was biphasic, with an early peak at 90 s and a secondary rise at 240 s, which persisted for 10 min. Natural human gastrin (sulfated and nonsulfated) and the active COOH-terminal octapeptide fragment of gastrin stimulated HIP, whereas the biologically inert NH2-terminal (1-13) fragment of gastrin had no effect. The activation of oxyntic cell HIP by gastrin was neutralized by an antiserum directed towards the COOH-terminus of gastrin and not by nonimmune serum. Cimetidine (10(-5) M) blocked 25% and atropine (10(-5) M) had no effect on gastrin-stimulated HIP. EGTA (10(-3) M) and LaCl3 (10(-3) M) inhibited the action of gastrin by 67 and 52%, respectively. The calmodulin antagonists, trifluoperazine (10(-5) M), pimozide (10(-5) M), and the naphthalene sulfonamides, W-7 and W-13 (10(-5) M), inhibited gastrin-stimulated HIP by 45.6 38.5, 42.3, and 37.2%, respectively. Higher doses of W-7 and W-13 (10(-4) M) inhibited gastrin-stimulated HIP by 83 and 67%. The Ca2+ ionophore, A23187 (10(-4) M), stimulated HIP. Thus, it appears that gastrin stimulation of HIP is complex. 25% of its action is via a histamine-dependent pathway. 45% of its action is dependent upon extracellular Ca2+. Its action is also in part dependent upon a Ca2+/calmodulin mechanism.

Cytochemical quantification of physiologic regulation of oxyntic cell carbonic anhydrase.

We have studied the physiologic factors regulating oxyntic cell activity using cytochemical quantification of carbonic anhydrase (CA) activity. Gastrin (10 (-16) to 10(-12)M), histamine (10(-17) to 10(-13) M), and carbamylcholine (10(-13) to 10(-8) M) caused a dose-dependent increase in CA in the oxyntic cells in guinea pig gastric fundus, maximal at 90 sec. The stimulation of CA by all three secretagogues was inhibited by the CA inhibitor, acetazolamide. The agonist activities were selectively blocked by respective antagonists. The benzimidazole derivative compound Hassle 149/94 (10(-3)M) abolished the actions of all agonists. Thus, histamine, gastrin and carbamylcholine have independent actions on oxyntic cell CA. The inhibition of the activity of all three secretagogues by H149/94 suggests a close link between CA activity and the functioning of the proton pump H+ + K+-ATPase.

Evidence for histamine H1 and H2 receptors in guinea-pig oxyntic cells.

We have previously shown that histamine in the dose range of 1.0 X 10(-16) to 1.0 X 10(-14) M caused a linear increase in oxyntic cell carbonic anhydrase (CA) activity. A maximal dose of the H2 antagonist, cimetidine, reduced the action of histamine by only 74 +/- 7.8% suggesting a possible H1 component of histamine action on oxyntic cells. Using quantitative cytochemistry of CA activity in guinea-pig oxyntic cells, we compared the dose-response relationships (1.0 X 10(-17)-1.0 X 10(-9) M) of specific H2 (dimaprit, 4-methylhistamine, impromidine) and H1 (2-pyridylethylamine) agonists with histamine (H1 and H2). In addition, we studied the effects of the specific H2 antagonist, cimetidine, and H1 antagonist, mepyramine, on histamine, dimaprit and 2-pyridylethylamine-stimulated CA activity. We found that guinea-pig oxyntic cells respond to both H1 and H2 agonists. H1 agonists were more potent but less efficacious than H2 agonists. H2 agonists were more efficacious, but less potent than H1 agonists. Cimetidine (10(-10)-10(-8) M) was a competitive antagonist of dimaprit-stimulated CA activity. However, at higher doses (10(-5) M), cimetidine antagonism of histamine-stimulated CA activity was of a mixed noncompetitive and competitive type. Cimetidine and mepyramine exhibited both H2 and H1 antagonism, but their affinities for their respective agonists were greater. The data suggests that the oxyntic cell has receptors for both H1 and H2 agonists. H1 agonism is important at low concentrations of agonists. The H1 and H2 receptor components appear to be functionally linked.