Scott J. Parkinson

Inhibition of Nitric Oxide Biosynthesis Promotes P-selectin Expression in Platelets Role of Protein Kinase C

Inhibition of NO synthesis promotes P-selectin expression on endothelial cells; however, the precise mechanism is unclear. Because No has been shown to inhibit protein kinase C (PKC) activity, we examined the hypothesis that the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) stimulates P-selectin expression on platelets via PKC activation. Ten-minute incubation with either phorbol 12-myristate 13-acetate (PMA), thrombin, or L-NAME significantly increased P-selectin expression on platelets (as assessed by flow-cytometric analysis) and PKC activity of platelet membranes. Increased P-selectin expression induced by either PMA, thrombin, or L-NAME was significantly attenuated by the selective PKC inhibitor UCN-01 (7-hydroxystaurosporine). Furthermore, L-NAME-induced P-selectin expression was significantly attenuated by either L-arginine, 8-bromo-cGMP, or sodium nitroprusside (SNP). Interestingly, L-NAME further potentiated P-selectin upregulation by thrombin. L-NAME, thrombin, and PMA also significantly increased polymorphonuclear leukocyte adherence to the coronary artery endothelium, an effect that was significantly attenuated by the anti-P-selectin monoclonal antibody PB1.3 or by UCN-01, L-arginine, 8-bromo-cGMP or SNP but not by D-arginine or he nonblocking anti-P-selectin monoclonal antibody NBP1.6. These results indicate that inhibition of NO synthesis induces rapid P-selectin expression, which appears to be at least partially mediated by PKC activation in platelets. Similar effects and mechanisms of L-NAME on P-selectin function were also observed in endothelial cells, another site of P-selectin expression.

Escherichia coli heat-stable enterotoxin receptors : a novel marker for colorectal tumors

PURPOSE: Receptors forEscherichia coliheat-stable toxin (ST) are selectively expressed in membranes of intestinal mucosa cells and colon carcinoma cellsin vitro,suggesting their use as a marker for colorectal tumorsin vivo.The present studies examined the expression and function of ST receptors in normal human tissues and primary and metastatic colorectal tumors obtained from patients at surgery. METHODS: Surgical specimens were obtained as follows: from normal colon; from primary adenocarcinomas from all anatomic divisions of the colon and rectum; from gallbladder, kidney, liver, lung, lymph node, ovary, peritoneum, stomach; and from colon carcinomas metastatic to liver, lung, lymph node, ovary, and peritoneum. Membranes prepared from these specimens were assessed for the presence and functional characteristics of ST receptors. RESULTS: ST bound specifically to membranes from each division of normal colon and rectum and all primary and metastatic colorectal tumors examined. The affinity and density of ST receptors were similar in tumors of different grades and from various metastatic sites. ST-receptor interaction was coupled to activation of guanylyl cyclase in all normal samples of colon and rectum and all primary and metastatic colorectal tumors examined. In contrast, neither ST binding nor ST activation of guanylyl cyclase was detected in any extraintestinal tissues examined. CONCLUSIONS: Functional ST receptors are expressed in normal colonic tissue and primary and metastatic colorectal tumors but not by extraintestinal tissues in humans. Expression of ST receptors does not vary as a function of the metastatic site or grade of these tumors. Receptors expressed by colorectal tumors retain their characteristic function, with binding of ST coupled to activation of guanylyl cyclase. These studies support the suggestion that ST receptors represent a specific marker for human colorectal tumors that may have use as a target for directing diagnostics and therapeutics to these tumorsin vivo.

Hypotensive Mechanisms of Amifostine

Amifostine, a chemo‐ and radioprotective agent developed as adjunctive therapy for malignancies, induces hypotension after ∼20% of patient administrations. This study examines the molecular mechanisms underlying hypotension induced by amifostine. Amifostine and its metabolite, WR‐1065, induced dose‐dependent hypotension in anesthetized rats that was not blocked by NG‐methyl L arginine (L‐NAME), an NO synthase inhibitor. WR‐1065 but not amifostine induced concentration‐dependent relaxation of isolated rat aortic rings in an endothelium‐independent fashion. Relaxation was not associated with increases in cGMP or cAMP and could not be blocked by L‐NAME or indomethacin. Similarly, neither amifostine or WR‐1065 activated adenylyl, particulate guanylyl, or soluble guanylyl cyclases. WR‐1065 relaxed rat aortic rings precontracted with norepinepherine, suggesting α‐adrenergic blocking activity. However, neither amifostine nor WR‐1065 altered the ability of prazosin or phentolamine to bind to α‐adrenergic receptors. Further, WR‐1065 had no effect on receptor‐mediated increases in intracellular calcium in BAL 17 murine B lymphocytes in vitro. Thus, hypotension after administration of amifostine is mediated by WR‐1065 and appears to result from direct relaxation of vascular smooth muscle. Smooth muscle relaxation induced by WR‐1065 is not related to production of nitric oxide, prostaglandins, or cyclic nucleotides; α‐adrenergic receptor antagonism; or interference with receptor‐dependent increases in intracellular calcium. Administration of ephedrine, an efficacious adrenergic agonist, attenuated hypotension induced by amifostine in anesthetized rats and may be useful in alleviating hypotension associated with amifostine administration in patients.

Interruption of transmembrane signaling as a novel antisecretory strategy to treat enterotoxigenic diarrhea

Bacteria that produce heat‐stable enterotoxins (STs), a leading cause of secretory diarrhea, are a major cause of morbidity and mortality worldwide. ST stimulates guanylyl cyclase C (GCC) and accumulation of intracellular cyclic GMP ([cGMP]i), which opens the cystic fibrosis transmembrane conductance regulator (CFTR)‐related chloride channel, triggering intestinal secretion. Although the signaling cascade mediating ST‐induced diarrhea is well characterized, antisecretory therapy targeting this pathway has not been developed. 2‐ChloroATP (2ClATP) and its cell‐permeant precursor, 2‐chloroadenosine (2ClAdo), disrupt ST‐dependent signaling in intestinal cells. However, whether the ability to disrupt guanylyl cyclase signaling translates into effective antisecretory therapy remains untested. In this study, the efficacy of 2ClAdo to prevent ST‐induced water secretion by human intestinal cells was examined. In Caco‐2 human intestinal cells, ST increased [cGMP]i, induced a chloride current, and stimulated net basolateral‐to‐apical water secretion. This effect on chloride current and water secretion was mimicked by the cell‐permeant analog of cGMP, 8‐bromo‐cGMP. Treatment of Caco‐2 cells with 2ClAdo prevented ST‐induced increases in [cGMP]i, chloride current and water secretion. Inhibition of the downstream consequences of ST‐GCC interaction reflects proximal disruption of cGMP production because 8‐bromo‐cGMP stimulated chloride current and water secretion in 2ClAdo‐treated cells. Thus, this study demonstrates that disruption of guanylyl cyclase signaling is an effective strategy for antisecretory therapy and provides the basis for developing mechanism‐based treatments for enterotoxigenic diarrhea.—Zhang, W., Mannan, I., Schulz, S., Parkinson, S. J., Alekseev, A. E., Gomez, L. A., Terzic, A., Waldman, S. A. Interruption of transmembrane signaling as a novel antisecretory strategy to treat enterotoxigenic diarrhea. FASEB J. 13, 913–922 (1999)

Interruption of Escherichia coli Heat-stable Enterotoxin-induced Guanylyl Cyclase Signaling and Associated Chloride Current in Human Intestinal Cells by 2-Chloroadenosine

Diarrhea induced by Escherichia coli heat-stable enterotoxin (STa) is mediated by a receptor guanylyl cyclase cascade. The present study establishes that an intracellular nucleotide-dependent pathway disrupts toxin-induced cyclic GMP (cGMP) production and the associated chloride (Cl−) flux that underlie intestinal secretion. Incubation of Caco 2 human intestinal epithelial cells with the nucleoside analog 2-chloroadenosine (2ClAdo) resulted in a concentration- and time-dependent inhibition of toxin-induced cGMP production. Inhibition of cGMP production correlated with the metabolic conversion of 2ClAdo to 2-chloroadenosine triphosphate. The effect of 2ClAdo did not reflect activation of adenosine receptors, inhibition of adenosine deaminase, or modification of the binding or distribution of STa receptors. Guanylyl cyclase activity in membranes prepared from 2ClAdo-treated cells was inhibited, in contrast to membranes from cells not exposed to 2ClAdo, demonstrating that inhibition of guanylyl cyclase C (GCC) was mediated by a noncompetitive mechanism. Treatment of Caco 2 cells with 2ClAdo also prevented STa-induced Cl− current. Application of 8-bromo-cGMP, the cell-permeant analog of cGMP, to 2ClAdo-treated cells reconstituted the Cl− current, demonstrating that inhibition of Cl− flux reflected selective disruption of ligand stimulation of GCC rather than the chloride channel itself. Thus, the components required for adenine nucleotide inhibition of GCC signaling are present in intact mammalian cells, establishing the utility of this pathway to elucidate the mechanisms regulating ST-dependent guanylyl cyclase signaling and intestinal fluid homeostasis. In addition, these data suggest that the adenine nucleotide inhibitory pathway may be a novel target to develop antisecretory therapy for enterotoxigenic diarrhea.

The E. Coli Heat‐Stable Enterotoxin (ST) Receptor is a Novel Marker for Targeting Therapeutics to Colon Carcinoma Cells

Clinical Pharmacology & Therapeutics (1996) 59, 187–187; doi: 10.1038/sj.clpt.1996.246

Adenine nucleotide-dependent inhibition of guanylyl cyclase C: Potential target for therapy of toxigenic diarrhea

Clinical Pharmacology & Therapeutics (1996) 59, 175–175; doi: 10.1038/sj.clpt.1996.199