Factors affecting the 16 alpha-hydroxylation of estrone 3-sulfate by guinea pig liver microsomes

The estrone 3-sulfate 16 alpha-hydroxylase of guinea pig liver microsomes has been demonstrated to be sensitive to CO. A CO/O2 ratio of 0.64 caused 50% inhibition of activity. Since inhibition was also obtained in the presence of 2-diethylaminoethyl-2,2-diphenylvalerate . HCl it seems likely that the hydroxylase is a cytochrome P450 containing system. A fourfold increase in enzyme activity was brought about by 40 mM Mg2+ or Ca2+ while the same concentration of Mn2+ resulted in a twofold increase. Lesser increases were seen with Na+ or K+ and complete inhibition was obtained in the presence of Fe2+, Cu2+, or EDTA. When assayed in the presence of detergent concentrations sufficiently small to guard against cytochrome P450 destruction, it was found that Cutscum, Triton X-100, and Triton N-101 each caused greatest inhibition of enzyme activity. Lesser inhibition was apparent in the presence of Miranol H2M, cholate, or deoxycholate. The nonionic detergent, Brij 35, caused least inhibition of all and, when hepatic microsomes were treated higher concentrations of Brij 35, about 80% of protein and over 95% cytochrome P450 were to be found in the 100 000 X g supernatant. Microsomal activity was more stable when stored at -20 degrees C in buffer containing glycerol, EDTA, and dithiothreitol than in buffer alone. Under best conditions only 10% of the hydroxylase activity was lost in one week.     

In vitro effect of calcitonin on guinea pig gallbladder

Calcitonin (CT) is a 32 amino acidic polypeptide hormone which has been found in almost all species and whose effects are mainly concerned with calcium and phosphorous homeostasis. Three preparations are employed for therapeutic uses: salmon (sCT), porcine (pCT) and human CT (hCT). The sCT is the most powerful one and in human volunteers a strong relaxing effect has been shown on gallbladder (GB) basal volume and emptying in response to a meal, intraduodenal instillation of a liquid meal and i.v. cholecystokinin (CCK) infusion. Our study was aimed at investigating if a direct sCT effect could be demonstrated on smooth muscle strips from guinea pig GBs "in vitro" (organ bath). Isometric contractions were measured in response to maximal doses of acetylcholine (ACh: 10(-4) M), KCl (80 mM) and cholecystokinin octapeptide (CCK-OP: 10(-6) M), in absence and in presence of four doses of sCT (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) and 1 x 10(-6) M). sCT did not affect the initial strip basal tone. ACh, CCK-OP and KCl caused, as expected, a powerful contraction of the strips, but no effect was shown when each of the sCT doses was administered before ACh (1.28+ 0.69 SEM without sCT vs 1.28g+ 0.69 with sCT; n = 6) and CCK-OP (1.46g+ 0.19 without sCT vs 1.46g+ 0.19 with sCT; n = 8) or 5 min after the induced KCl contraction. On the basis of these preliminary results, we conclude that no evidence of a direct sCT effect was found on guinea pig GBs when considering either basal smooth muscle tone or isometric contraction in response to ACh, KCl and CCK-OP. Further studies are therefore required to clarify the influence of CT on GB dynamics in vivo and to elucidate its the physiological significance.     

Choline glycerophospholipid biosynthesis in the guinea pig heart

The aims of this study were to (i) elucidate the biosynthetic pathways for the formation of plasmenylcholine in the mammalian heart and (ii) investigate whether the control of choline glycerophospholipid production is different in hearts with high plasmenylcholine content. Guinea pig hearts were used throughout this study, since 34% of the cardiac choline glycerophospholipids in this species is present in the plasmenylcholine form. By perfusion of the guinea pig heart in the Langendorff mode with labeled choline, we demonstrated that the majority of plasmenylcholine in the heart was synthesized via the CDP-choline pathway. The ability of the heart to form plasmenylcholine from CDP-choline and 1-alkenyl-2-acylglycerol was also shown. We postulate that 1-alkenyl-2-acylglycerol in the guinea pig heart might originate from the hydrolysis of plasmenylethanolamine. In mammalian liver and other tissues, the CDP-choline pathway is the major pathway for phosphatidylcholine biosynthesis and the rate-limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase. The results obtained from the present study support this supposition. In addition, evidence was obtained indicating that phosphorylation of choline by choline kinase in the CDP-choline pathway may also be rate limiting. Although the involvement of choline kinase as a rate-limiting enzyme in the CDP-choline pathway has been shown in a number of cell cultures, the rate-limiting role of this enzyme in intact mammalian organs has not been previously reported. The rationale for the presence of more than one rate-limiting step in the CDP-choline pathway in the guinea pig heart remains undefined.     

3d Mechanical properties of the partially obstructed guinea pig small intestine

Background and aimsPartial obstruction of the small intestine results in severe hypertrophy of smooth muscle cells, dilatation and functional denervation. Hypertrophy of the small intestine is associated with alteration of the wall structure and the mechanical properties. The aims of this study were to determine three dimensional material properties of the obstructed small intestine in guinea pigs and to obtain the 3D stress–strain distributions in the small intestinal wall.MethodsPartial obstruction of mid-jejunum was created surgically in five guinea pigs that were euthanized 2 weeks after the surgery. Ten-cm-long segments proximal to the obstruction site were used for the stretch-inflation mechanical test using a tri-axial test machine. The outer diameter, longitudinal force and the luminal pressure during the test were recorded simultaneously. An anisotropic exponential pseudo-strain energy density function was used as the constitutive equation to fit the experimental loading curve and for computation of the stress–strain distribution.ResultsThe wall thickness and the wall area increased significantly in the obstructed jejunum (P<0.001). The pressure—outer radius curves in the obstructed segments were translated to the left of the normal segments, indicating wall stiffening after the obstruction. The circumferential stress and the longitudinal stress through the wall were higher in the obstructed segments (P<0.02). This was independent of whether the zero-stress state or the no-load states were used as the reference state.ConclusionThe mechanical behaviour of the obstructed small intestine can be described using a 3D constitutive model. The obstruction-induced biomechanical properties change was characterized by higher circumferential and longitudinal stresses in the wall and altered material constants in the 3D constitutive model.     

Removal from the small intestine of tubercle bacilli by macrophages in the guinea pig

Summary The present study was arranged to test the reaction of the intestinal cell barrier toward the presence of chromogenic, acid-fast bacteria. The bacilli were introduced into the alimentary tract through a modified stomach tube. It was found that these bacterial forms were transported by macrophages through the columnar epithelium, within one to two hours following the test feeding. No other cellular elements such as neutrophils or columnar epithelial cells were observed in association with this process.Supported by a grant from the Nebraska Heart Association. Department of Zoology, University of Nebraska, Contribution No. 418.     

Epithelial distribution of neural receptors in the guinea pig small intestine

Neural and paracrine agents, such as dopamine, epinephrine, and histamine, affect intestinal epithelial function, but it is unclear if these agents act on receptors directly at the enterocyte level. The cellular localization and villus-crypt distribution of adrenergic, dopamine, and histamine receptors within the intestinal epithelium is obscure and needs to be identified. Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs. Enterocytes were separated from intraepithelial lymphocytes by flow cytometry and specific binding was determined using fluorescent probes. Alpha1-adrenergic receptors were located on villus and crypt intraepithelial lymphocytes and enterocytes. Beta-adrenergic receptors were found on villus and crypt enterocytes. Dopamine receptors were found on all cell types examined, whereas histamine receptors were not detected (<10% for each cell population). These studies demonstrated that (1) receptors for epinephrine and dopamine exist on epithelial cells of the guinea pig jejunum, (2) beta-adrenergic receptors are found primarily on villus and crypt enterocytes and (3) intraepithelial lymphocytes contain alpha1-adrenergic, but have few beta-adrenergic, receptors. The presence of neural receptors suggests that these agents are acting, at least in part, at the enterocyte or intraepithelial lymphocyte levels to modulate intestinal and immune function.     

Mechanogram profile of the guinea pig intestine. The supertone

The authors detected the possibility, during the phase of low tone contraction of isolated intestine, to develop a further contraction, a supertone (S), simply washing the preparation. It has been evidenced that S appears when the tonic phase is low; it does not disappear if the tone is increased lowering NaCl concentration in the saline plasma; is absent in the KCl induced contraction; is induced also by low doses of atropine; is decreased by digitalic agents. The results obtained lead the authors to conclude that: the reduced tonic phase is due to a positive factor, the Ca++ of the cytoplasm, and to a negative factor, the high level of cytoplasmic Na+. This high level of Na+ is maintained by a dynamic equilibrium with ion input through channels opened by muscarinic agent and output by a Na-K-ATP asi pump. The Na+ output is predominant and fraction of Ca++, no longer counterbalanced by Na+, causes the rise of S.     

Glucagon-like peptide-1 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro

Glucagon-like peptide-1 (GLP-1) acts at the G protein-coupled receptor, GLP-1R, to stimulate secretion of insulin and to inhibit secretion of glucagon and gastric acid. Involvement in mucosal secretory physiology has received negligible attention. We aimed to study involvement of GLP-1 in mucosal chloride secretion in the small intestine. Ussing chamber methods, in concert with transmural electrical field stimulation (EFS), were used to study actions on neurogenic chloride secretion. ELISA was used to study GLP-1R effects on neural release of acetylcholine (ACh). Intramural localization of GLP-1R was assessed with immunohistochemistry. Application of GLP-1 to serosal or mucosal sides of flat-sheet preparations in Ussing chambers did not change baseline short-circuit current (I(sc)), which served as a marker for chloride secretion. Transmural EFS evoked neurally mediated biphasic increases in I(sc) that had an initial spike-like rising phase followed by a sustained plateau-like phase. Blockade of the EFS-evoked responses by tetrodotoxin indicated that the responses were neurally mediated. Application of GLP-1 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-1 receptor antagonist exendin-(9-39) suppressed this action of GLP-1. The GLP-1 inhibitory action on EFS-evoked responses persisted in the presence of nicotinic or vasoactive intestinal peptide receptor antagonists but not in the presence of a muscarinic receptor antagonist. GLP-1 significantly reduced EFS-evoked ACh release. In the submucosal plexus, GLP-1R immunoreactivity (IR) was expressed by choline acetyltransferase-IR neurons, neuropeptide Y-IR neurons, somatostatin-IR neurons, and vasoactive intestinal peptide-IR neurons. Our results suggest that GLP-1R is expressed in guinea pig submucosal neurons and that its activation leads to a decrease in neurally evoked chloride secretion by suppressing release of ACh at neuroepithelial junctions in the enteric neural networks that control secretomotor functions.     

Lipid biosynthesis in liver slices of the foetal guinea pig.

Lipid synthesis as measured by the incorporation of acetate or 3H2O into slices of foetal liver, is much higher than in slices of adult liver and shows a peak at about two-thirds of gestation. At this time the synthesis from glucose was low and reached a peak 10 days later. The changes in the activity of ATP citrate lyase, which mirrored acetate incorporation, and the effect of glucose and pyruvate on acetate corporation into lipid suggests that some of the lipid synthesis occurs via intramitochondrial acetyl-CoA production from acetate. Despite this, lipid synthesis was not inhibited by (-)-hydroxycitrate. The low rate of synthesis from glucose at two-thirds of gestation is ascribed to the low activity of pyruvate carboxylase at this time and a role for a phosphoenolpyruvate carboxykinase in providing oxaloacetate for lipogenesis is proposed. The activity of fatty acid synthetase broadly agreed with the changes in lipid synthesis, whereas the activity of acetyl-CoA carboxylase was barely sufficient to account for the rates of lipid synthesis in vivo. Acetate and short-chain fatty acids are likely to be the major precursors for lipid synthesis in vivo.     

Peptide YY induces nerve-mediated responses in the guinea pig intestine.

The actions of peptide YY (PYY) were studied in longitudinal organ-bath preparations of the guinea pig intestine. PYY induced concentration-dependent (10(-9)-5 x 10(-8) M) relaxations of tissue from the duodenum, jejunum, ileum, and colon. These responses were unaffected by adrenergic blockade and atropine treatment but could be prevented by tetrodotoxin. The pharmacology of PYY actions in segments of the small and large intestine indicated the involvement of intrinsic nonadrenergic, noncholinergic inhibitory neurones in the relaxation response to this peptide. All tissues could be made tachyphylactic to PYY without affecting their ability to respond to the direct acting muscle relaxants ATP or papaverine. Moreover, nicotinic ganglion stimulated relaxations and cholinergic nerve-mediated contractions were also unaffected. These results show applied PYY to have potent neurogenic actions in the guinea pig intestine with some similarities to PYY actions in the rat intestine.     

Presence of atrial natriuretic peptide-like material in guinea pig intestine

Acidic extracts of guinea pig jejunum and colon contain atrial natriuretic peptide-like material (IR-ANP) detected by radioimmunoassay after purification by Sephadex G-50 gel filtration. Immunohistochemical analysis of guinea pig colon also revealed IR-ANP to be located directly beneath the lamina muscularis mucosae. High performance gel permeation chromatography (HP-GPC) and reverse phase high performance chromatography (RP-HPLC) of the IR-ANP showed correspondence to the 15 kD ANP precursor molecule (pro-ANP). No low molecular weight forms of ANP were detected. The extracted pro-ANP could be converted to alpha-ANP-like material by incubation with serum or supernatant of colonic homogenate. These data indicate the intestine to be a further site of ANP synthesis.     

In vitro production of prostaglandins E and F by the guinea pig ovarian tissue

The ability of guinea pig ovarian tissue to biosynthesize prostaglandins E and F from endogenous precursors has been investigated in vitro. Estimations of prostaglandins were carried out using a sensitive radioimmuno assay during seven days preceding, and up to one day, following oestrous. Prostaglandins E and F were present in the ovarian tissue throughout the period investigated. Prostaglandin concentrations in samples incubated without enzymic inhibition were significantly higher than in samples incubated after enzymic inhibition with ethanol. This indicates that guinea pig ovarian tissue is able to synthesize prostaglandins from endogenous precursors.     

High-affinity 3H-substance P binding to longitudinal muscle membranes of the guinea pig small intestine

The binding of 3H-substance P (3H-SP) to longitudinal muscle membranes of the guinea pig small intestine has been characterized. The binding of 3H-SP exhibited a high affinity (Kd = 0.5nM). It was saturable (Bmax = 2 fmoles/mg tissue), reversible, and temperature-dependent. Kinetic studies and competition of 3H-SP binding by unlabeled SP yielded Kd and Ki values, respectively, which were in good agreement with the Kd calculated from saturation studies. The binding of 3H-SP appeared to be dependent on the presence of divalent cations in the incubation buffer. It was displaced by SP and various analogs and fragments in the rank order of SP greater than SP-(2-11) = SP-(3-11) greater than Nle11- SP = physalaemin greater than SP-(4-11) greater than SP-(5-11) greater than eledoisin much greater than SP-(7-11). Our results indicate that 3H-SP binds in longitudinal muscle of the guinea pig small intestine to a biologically relevant receptor which in many respects resembles the SP receptor characterized in the brain and the salivary gland of the rat.     

ATP-induced currents in submucous plexus neurons of the guinea pig small intestine

ATP-induced membrane durrents in the submucous neurons of the guinea pig small intestine were studied using the whole-cell patch-clamp recording technique. Being applied at –50 mV. ATP activated an inward non-selective cationic current in 68.3% of the investigated neurons. An increase in ATP concentration within the 1–1,000 µM range resulted in the s-like increase in the amplitude of ATP-induced current. The EC₅₀ was 150.0±18.5 µM, while the Hill number was 1.6. The current was selectively activated by ATP and was not blocked by P2 purinoreceptor antagonist suramin (50–300 µM).,-Methylene-ATP (100–200 µM) and,-methylene-ATP (100–200µM), which are P2-purinoreceptor agonists, as well as adenosine (100–300 µM), exerted no effects. Reactive blue 2, if applied up to 4 min, enhanced ATP-induced current, while its longer application partially suppressed this current. In most submucous neurons, acetylcholine (ACh) likewise activated an inward cationic current. The amplitude of ACh-induced current was lower if ACh was applied during a long-lasting application of ATP than if ACh only was applied. Hexamethonium (50 µM), d-tubocurarine (20–40 µM), and trimethaphan (30 µM) completely and reversibly blocked ACh-induced currents, regardless of the presence of ATP, and did not affect ATP-induced currents. The results suggest that ATP-induced currents in submucous neurons are due to activation of a unique type of P2 purinoreceptors, which function in connection with nicotinic ACh receptors.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 100–110, March–June, 1996.     

The villi contribute to the mechanics in the guinea pig small intestine

Previous studies have shown that intestinal mucosa is compressed in vivo. The present study investigated the contribution of the mucosal villi to the biomechanical properties in circumferential direction in the guinea pig jejunum. Eight 20-cm-long jejunal segments were excised and each separated into two 10-cm-long segments. The mucosal villi were scraped off from half the segments. The segments were pressurized in vitro with Krebs solution from 0-10cmH(2)O using a ramp distension protocol with simultaneous diameter recordings. Circumferential stresses and strains were computed from the diameter, pressure and the zero-stress state data. Removing the villi resulted in small opening angles (139+/-16 degrees vs 189+/-27 degrees with villi) and small absolute values of residual strain (inner: -0.05+/-0.03 vs -0.33+/-0.06 with villi; outer: 0.11+/-0.04 vs 0.33+/-0.08 with villi) (P<0.001). The outer diameter as a function of the pressure did not differ between jejunal segments with villi and without villi. The average mid-wall stress-strain curve without villi was shifted to the left compared to the segment with villi, indicating the wall was stiffer without villi. However, if the stress-strain computation for the segments with villi was referenced to the zero-stress state of the segments without villi, the curve was only partly shifted to the left. In conclusion, this paper provides the first direct experimental evidence that the villi are important for the biomechanical properties of guinea pig small intestine in circumferential direction, because the villi not only affect the zero-stress state configuration but also partially affect the stress-strain distribution in the intestinal wall. Therefore, the villi should be taken into account in the analysis of biomechanical properties of the intestinal wall.