Reisuke Saito

Bile Salt Stimulation of Colonic Epithelial Proliferation Evidence for Involvement of Lipoxygenase Products

Prostaglandin E2 (PGE2) and several other prostaglandins synthesized by colon suppress the proliferative activity of colonic epithelium. However, bile salts stimulate colonic epithelial proliferation despite the actions of bile salts to enhance the release of arachidonate and consequent colonic synthesis of PGE2. The current study was conducted to assess whether bile salt-induced increases in colonic formation of arachidonate metabolites other than PGE2 were linked to the stimulation of the proliferative activity of colonic epithelium. Within 10 min of addition, deoxycholate markedly stimulated the in vitro release of [14C]arachidonate from prelabeled rat colon. When given in vivo by intracolonic instillation deoxycholate (10 mumol) increased colonic accumulation of immunoreactive prostaglandin E (PGE), thromboxane B2 (TXB2), and the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) by two to fourfold over control in 30 min. This effect of intracolonic deoxycholate was followed by a ninefold increase in mucosal ornithine decarboxylase activity (4 h), and a subsequent two to threefold increase in [3H]thymidine [( 3H]Thd) incorporation into DNA of either mucosal scrapings or isolated pools of proliferative colonic epithelial cells (24 h). Intracolonic instillation of indomethacin (50 mumol) suppressed to low or undetectable levels both basal colonic accumulation of PGE and TXB2 and the increases in each parameter induced by subsequent instillation of deoxycholate. By contrast, indomethacin enhanced accumulation of 12-HETE in both control colons and those subsequently exposed to deoxycholate. The increases in 12-HETE induced by indomethacin alone were correlated with stimulation of mucosal ornithine decarboxylase activity and [3H]Thd incorporation into mucosal DNA. Indomethacin also enhanced the increases in these parameters induced by deoxycholate. Intracolonic instillation of phenidone (25-100 mumol) suppressed accumulation of PGE, TXB2, and 12-HETE in control colons and the increases in these parameters induced by a subsequent instillation of deoxycholate. Phenidone alone did not alter mucosal ornithine decarboxylase activity or [3H]thymidine incorporation into mucosal DNA. However, phenidone suppressed or abolished increases in these parameters induced by a subsequent instillation of deoxycholate. 4-(2-[IH-imidazol-1-yl]ethoxy) benzoic acid hydrochloride UK 37,248, which selectively reduced colonic TXB2 to undetectable levels without altering PGE or 12-HETE, had no effect on control or deoxycholate-induced increases in mucosal ornithine decarboxylase activity or [3H]Thd incorporation into DNA. Neither indomethacin nor phenidone altered the increases in [(14)C]arachidonate release induced in vitro by deoxycholate. Chenodeoxycholate and cholate also stimulated [(14)C]arachidonate release from colon in vitro within 10 min, and increased colonic 12-HETE (30 min) and mucosal ornithine decarboxylase activity (4 h) upon intracolonic installation. Prior installation of phenidone inhibited the increases in both 12-HETE and ornithine decarboxylase activity induced by these bile salts. The results support a role for bile salt-induced increases in colonic accumulation of lipoxygenase products, as reflected by 12-HETE, in the subsequent stimulation of the proliferative activity of colonic epithelium.

Role of local prostaglandin synthesis in the modulation of proliferative activity of rat colonic epithelium.

The role of local prostaglandin (PG) synthesis in the modulation of the proliferative activity of colonic epithelium was examined in rat colon. Experimental rats were given either indomethacin (5 mg/kg s.c. every 8 h for three doses) or aspirin (0.5 g/100 g diet for 3 d). In rats treated with indomethacin or aspirin, the incorporation of [3H]thymidine (dThd) into DNA in vivo was increased approximately twofold over control in mucosal scrapings from distal colon, and approximately threefold over control in the proliferating pool of epithelial cells isolated from distal colon. [3H]dThd incorporation into DNA was also examined ex vivo immediately after distal colonic resection. It was approximately twofold higher in mucosa of colonic segments (1-h incubation) from rats treated with indomethacin or aspirin in vivo, compared with corresponding values of segments from control rats. Immunoreactive (i) prostaglandin E (PGE), the dominant PG product of colon segment incubates by high-performance liquid chromatography analysis of [14C]arachidonate metabolites, was markedly (95%) reduced in the media of 1-h colon incubates from indomethacin- or aspirin-treated rats, compared with control rats. Moreover, the cyclic (c)AMP content of mucosa of segments from indomethacin- or aspirin-treated rats was significantly lower than that of control rats. Prolonged incubation (4-24 h) of colonic segments from indomethacin-treated rats, in the absence of indomethacin in vitro, led to an eventual return of [3H]dThd incorporation into DNA, iPGE, and mucosal cAMP to control values. Conversely, inclusion of indomethacin (0.25 mM) in the incubations (6 h) of colonic segments from indomethacin-treated rats resulted in persistent suppression of iPGE and mucosal cAMP, as well as persistent enhancement of [3H]dThd incorporation into mucosal DNA. However, incubation of colonic segments from control rats (no in vivo drug exposure) with indomethacin or aspirin in vitro for periods up to 24 h failed to alter DNA synthesis, despite marked reduction in media iPGE and lower mucosal cAMP. The latter observations suggested that additional in vivo factors initiated the enhancement of DNA synthesis in indomethacin- or aspirin-treated rats. Exogenous PGE2, D2, I2, or F2 alpha, each of which increased the endogenous mucosal cAMP content of incubated colonic segments from control, indomethacin- or aspirin-treated rats, all suppressed [3H]dThd incorporation into mucosal DNA in vitro. Dibutyryl cAMP, but not dibutyryl cGMP, had an analogous suppressive effect on in vitro [3H]dThd incorporation into DNA. Thus, the present observations are consistent with an inhibitory action of endogenous colonic PG synthesis on the proliferative activity of colonic epithelium. This action may be mediated through cAMP.

Cardiopulmonary bypass after prolonged cardiac arrest in dogs

Ventricular fibrillation (VF) cardiac arrest of more than ten minutes can be survived by cerebral neurons, but restoration of spontaneous circulation (ROSC) by external CPR is unreliable. Cardiopulmonary bypass (CPB) permits control of pressure, flow, oxygenation, temperature, and composition of blood. After 12 1/2 minutes of normothermic VF cardiac arrest, CPB was used as a research tool for reperfusion and assisted circulation for two hours in ten dogs without thoracotomy, with plasma substitute priming, and without preceding CPR (a deliberately nonclinical scenario). Recovery was compared with that in ten control dogs in which standard CPR with advanced life support (ALS) for up to 30 minutes was used to achieve ROSC. Both groups subsequently had blood pressure, blood gases, ventilation, and other parameters controlled for 20 hours, and intensive therapy to 72 hours. CPB achieved ROSC more successfully (ten of ten vs six of ten controls) (P less than .05), and more rapidly, with fewer defibrillation attempts and with less epinephrine (P less than .05). CPB improved 72-hour survival (seven of ten vs two of ten controls) (P = .025). Between two and 24 hours, of those with ROSC, intractable cardiogenic shock killed four of six control dogs (NS). CPB was followed by fewer arrhythmias. CPB increased recovery of consciousness (five of ten CPB vs zero of six controls with ROSC) (P = .037), but achieved neurologic normality in only one of ten. Cardiac arrest and CPB (without CPR) resulted in less myocardial morphologic damage than did standard CPR (P less than .025).(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of N‐methyl‐N′–nitro‐N‐nitrosoguanidine action on the guanylate cyclase‐guanosine 3′5′ monophosphate system of isolated colonic epithelial cells

The effects of the colon carcinogen N‐methyl‐N′–nitro‐N‐nitrosoguanidine (MNNG) on the guanylate cyclase (GC)‐guanosine 3′5′ monophosphate (cGMP) system were examined in epithelial cells isolated from the distal colon of the rabbit. MNNG (1 μM to 1 mM) increased cGMP accumulation 2‐ to 50‐fold within 5 minutes, without producing concurrent increases in adenosine 3′5′ monophosphate. The action of MNNG to increase cGMP was expressed in the absence of both extracellular calcium and molecular O2. cGMP was also increased by nitrite and by nitric oxide (NO), a reactive derivative of MNNG and nitrite. Increases in cGMP induced by MNNG in colonic epithelial cells were suppressed by retinol and butylated hydroxyanisole (BHA), whereas the effect of a submaximal concentration of MNNG was potentiated by 0.1 mM deoxycholate. MNNG, nitrite, and NO activated GC in the 100,000 × g particulate fraction, the predominant form of the enzyme in colonic epithelial cells. However, this action was expressed only in the presence of an appropriate concentration of a reducing agent (dithiothreitol, glutathione or ascorbate), which concurrently led to formation of a paramagnetic complex with the ESR spectral characteristics of NO‐heme in colonic particulate fractions exposed to MNNG. By contrast, several preformed paramagnetic NO‐hemeprotein complex (NO‐hemoglobin, NO‐catalase and NO‐cytochrome P‐450) increased particulate GC of colonic epithelial cells in the absence of added co‐factors. These observations suggest that formation of NO‐heme may be involved in the process by which MNNG activates particulate GC of colonic epithelium. Retinol, BHA, high concentrations of dithiothreitol, and the thiol blocker N‐ethylmaleimide suppress the effects of both MNNG and of preformed NO‐heme complexes to activate GC. Thus, the effects of these inhibitors may be expressed, at least in part, at point(s) in the enzyme activation pathway distal to generation of NO‐heme from MNNG. Stimulation of the GC system of colonic epithelium by MNNG, as well as the inhibition of this effect by antioxidants (BHA and retinol) and its potentiation by a putative co‐carcinogen (deoxycholate) could play a role in the expression of the oncogenic actions of MNNG in colon.