Localization of cholesterol in the colonic epithelium of the guinea pig: regional differences and functional implications

Summary It is generally accepted that variations in membrane cholesterol content affect the fluidity of the bilayer, thus altering its permeability. In the biological membranes, in physiological conditions, a high cholesterol content rigidifies the bilayer decreasing its permeability, a lower cholesterol content induces the opposite effect by increasing the permeability. Since differences in the epithelial permeability for short chain fatty acids have previously been demonstrated in the proximal and distal colon of the guinea pig, these two regions were investigated to establish whether differences in membrane cholesterol content of the absorbing cells can be demonstrated. Freeze-fracture replicas of filipin-treated colonic tissue were used. The results show that in the proximal colon the density of filipin cholesterol complexes located on the luminal plasma membrane of the columnar absorbing cells was significantly higher (about twice) than in the distal colon. Therefore the lower amount of cholesterol present in the membrane of the absorbing cells in the distal colon indicates a greater fluidity of the membranes of the epithelial cells in this region. Such fluidity could be correlated to the higher absorption rates of shortchain fatty acids characteristic of this region.     

Further evidence for non-pancreatic insulin immunoreactivity in guinea pig brain

The existence of large amounts of insulin in rat brain and of a porcine- or rat-like insulin in guinea pig brain have been disputed on the basis of differing results from standard (Method I) and hydrophobic adsorption techniques (Method II) for concentrating insulin from acid ethanol extracts. To try to resolve these differences, acid ethanol extracts of rat and guinea pig brains were divided into equal aliquots and concentrated for insulin radioimmunoassay (RIA) by both techniques. The RIA used guinea pig anti-porcine insulin serum, with 50% B0 for purified pancreatic porcine, rat and guinea pig insulin standards being 1.35, 2.38 and greater than 1,000 ng/ml, respectively. Oral glucose (4 g/kg) produced plasma glucose of 377 mg/dl in a guinea pig by 20 min but was not associated with any porcine- or rat-like immunoreactive insulin. Dilutions of guinea pig and rat brain extracts had parallel cross-reactivity with insulin standard curves. Insulin contents of rat brain (uncorrected for recovery) against porcine and rat insulin standards, respectively, were 1.33 and 1.93 ng/g (Method I) and 5.93 and 11.67 ng/g (Method II). Rat plasma was 0.85 and 1.42 ng/ml, respectively. Guinea pig contained 1.35 and 1.89 ng/g (uncorrected), respectively (Method I), and 2.99 and 5.62 ng/g, respectively (Method II). Guinea pig plasma was below the sensitivity of the RIA (less than 0.15 ng/ml). These results suggest that a porcine- or rat-like insulin may exist in guinea pig brain.     

Mechanism and localization of cardiolipin biosynthesis revisited: evidence for the identical mechanism and different localization in mitochondrial and submitochondrial membranes isolated from guinea pig and rat liver

The mechanism of cardiolipin (diphosphatidylglycerol) biosynthesis was examined in mitochondria and outer and inner mitochondrial membranes prepared from guinea pig and rat livers to determine whether this formation from phosphatidylglycerol was absolutely dependent on cytidinediphosphodiglyceride, as previously reported for intact mitochondria. Experimental results confirmed that the biosynthesis of cardiolipin, from the membrane-bound radioactive phosphatidylglycerol in intact mitochondria isolated from guinea pig and rat liver, was absolutely dependent on CDP-diglycerides and required the addition of divalent cations. Furthermore, the same mechanism for the biosynthesis of cardiolipin was operational in the outer and inner mitochondrial membranes. This biosynthesis was associated with both the outer and inner mitochondrial membranes prepared from guinea pig liver, but only with the inner mitochondrial membranes prepared from rat liver. The release of radioactive glycerol was also measured, but the amount obtained did not satisfy the stoichiometric requirement for CDP-diglyceride-independent biosynthesis of cardiolipin from 2 mol of phosphatidylglycerol with the liberation of 1 mol of glycerol. Therefore, it was concluded that this mechanism is not involved in the biosynthesis of cardiolipin in mitochondrial and submitochondrial membranes prepared from guinea pig and rat liver.     

Bradykinin receptors: functional similarities in guinea pig gut muscle and mucosa

It is well established that bradykinin can stimulate mucosal electrolyte transport. However, the receptor type which mediates this effect has not been fully characterized. Recent studies have suggested that bradykinin and related kinins may act at two types of receptors designated as B1 and B2. We have determined the effect of bradykinin on electrolyte secretion across guinea pig ileal mucosa and longitudinal muscle in vitro in the presence and absence of D-Phe7-bradykinin (B2 antagonist) and des-Arg9-(Leu8)-bradykinin (B1 antagonist). The B2 antagonist (less than 100 microM) did not affect resting muscle tension or basal electrolyte transport but at 6-30 microM it caused a parallel rightward shift in the concentration-response curves to bradykinin in the mucosa (Ki = 4 microM) and muscle (Ki = 6 microM). Changes in electrolyte transport and muscle contractility evoked by bethanechol and substance P were not affected by the B2 antagonist (30 microM) in either the muscle or the mucosa. Moreover, changes in electrolyte transport and muscle contractility produced by bradykinin were not altered by the B1 antagonist (30 microM). Finally, the B1 agonist des-Arg9-bradykinin (10 nM-1 microM) was not active in either preparation. These data suggest that under normal conditions, ileal secretion and smooth muscle contractility in the guinea pig are regulated by B2-type bradykinin receptors.     

Alternative mRNA splice variants of the rat ClC-2 chloride channel gene are expressed in lung: genomic sequence and organization of ClC-2.

The ClC-2 epithelial cell chloride channel is a voltage-, tonicity- and pH-regulated member of the ClC super family. We have previously shown that rat lung ClC-2 (rClC-2) is down-regulated at birth, and molecular diversity is generated by alternative splicing [Murray et al. (1995) Am. J. Respir. Cell Mol. Biol. 12, 597-604; Murray et al. (1996) Am. J. Physiol. 271, L829-L837; Chu et al . (1996) Nucleic Acids Res. 24, 3453-3457]. To investigate other possible mRNA splice variations, we sequenced the entire rClC-2 gene and found that ClC-2Sa (formerly ClC-2S) results from the deletion of exon 20. The preceding intron 19 has an unusually high CT content and a rare AAG acceptor site. Because both features were also found in intron 13, we next tested the hypothesis that intron 13 would be involved in alternative splicing. As predicted, a second splice product, ClC-2Sb, was found by RT-PCR, but only in lung. When we compared the genomic maps of rClC-2 and human ClC-1 (hClC-1), striking similarities were found in each exon except for rClC-2 exon 20, which is absent in hClC-1. These observations suggest that ClC-1 and ClC-2 may have evolved by gene duplication, mutation and DNA rearrangement.     

The submitochondrial localization adenylate kinase: an enzymatic marker for the inner surface of the outer membrane of lung mitochondria in guinea pig

Guinea pig lung mitochondrial adenylate kinase activity was measured under isotonic and hypotonic conditions. The activity differed in sensitivity to trypsin. Under isotonic conditions, the enzyme resisted the action of trypsin, where as the enzyme was destroyed substantially by trypsin under hypotonic conditions.     

The nature of 2-locus epistatic interactions in animals: evidence from Sewall Wright's guinea pig data

Summary The nature of epistatic interactions affects covariance between relatives and the expression of heterosis in various crossbred genotypes. The investigation of these interactions for metric traits requires large data sets of a suitable type. Data from Sewall Wright's early work with guinea pigs are used to compare the goodness-of-fit of seven biological models of 2-locus interaction for the six out of eleven traits in which epistatic effects are apparent. The model equivalent to additive x additive epistasis gives the best general fit over traits, with an average transformed R² value significantly greater than that of the next best fitting model (P<0.05). This result is compatible with results from the one other study in this area, using data from mice. It is concluded that, based on results available to date, the additive x additive 2-locus model of epistatic interaction appears most suitable for reduced genetic models.     

Further evidence for non-T-cell regulation of delayed hypersensitivity in the guinea pig.

The nature of the suppressor activity in the spleens of guinea pigs immunized with dinitrophenyl-bovine γ-globulin in Freund's incomplete adjuvant was investigated. An anti-T-cell serum was prepared in rabbits and, after extensive absorption, showed specific killing for T-lymphocytes. After treatment with this antiserum and complement, spleen cells from animals immunized with the antigen in Freund's complete adjuvant showed marked reduction in ability to transfer sensitivity to normal recipients. However, when immune spleen cells, treated in the same way, were transferred into antigen immunized animals which had been pretreated with cyclophosphamide, the suppressor activity was unaltered. These results confirm earlier impressions that the regulation of delayed hypersensitivity reactions in the guinea pig is normally mediated by non-T-cells.     

Neuropeptides in guinea pig trachea: Distribution and evidence for the release of CGRP into tracheal lumen

The airways of the guinea pig are richly innervated by peptide-containing nerve fibers. Among the most abundant neuropeptides are calcitonin gene-related peptide (CGRP) and substance P (SP), which are stored in nerve fibers located predominantly within and beneath the epithelium, and vasoactive intestinal peptide (VIP), which is located in fibers running mainly among smooth muscle bundles and seromucous glands. Sensory denervation (capsaicin treatment) of adult guinea pigs caused an almost total disappearance of CGRP- and SP-containing nerve fibers, while the density of VIP-containing nerve fibers located in smooth muscle seemed to increase. In the isolated trachea, perfused luminally, CGRP was found to appear in the intraluminal fluid after exposure to capsaicin but not after electrical vagal stimulation. CGRP concentrations in the tracheal wall did not change significantly. Luminally applied CGRP did not affect smooth muscle tension, measured as intraluminal volume changes.     

Ultrastructural evidence for adrenergic nerve degeneration in the guinea pig uterus during pregnancy

Summary In the guinea pig myometrium, the adrenergic nerves selectively demonstrated at the ultrastructural level after treatment with 5-OH-DA, show varying degree of degeneration during pregnancy. The changes are more extensive in a late gestational stage (40–45 days) than in an early one (20–25 days), and are particularly evident in the uterus overlying the conceptus as compared to the regions between the fetuses. Scattered degenerative changes were also observed in myometrial specimens from virgin animals, but probably reflect the normal continuous turnover of axons.     

Stretch-activated neuronal pathways to longitudinal and circular muscle in guinea pig distal colon

The role of the longitudinal muscle (LM) layer during the peristaltic reflex in the small and large intestine is unclear. In this study, we have made double and quadruple simultaneous intracellular recordings from LM and circular muscle (CM) cells of guinea pig distal colon to correlate the electrical activities in the two different muscle layers during circumferential stretch. Simultaneous recordings from LM and CM cells (<200 microm apart) at the oral region of the colon showed that excitatory junction potentials (EJPs) discharged synchronously in both muscle layers for periods of up to 6 h. Similarly, at the anal region of the colon, inhibitory junction potentials (IJPs) discharged synchronously in the two muscle layers. Quadruple recordings from LM and CM orally at the same time as from the LM and CM anally revealed that IJPs occurred synchronously in the LM and CM anally at the same time as EJPs in LM and CM located 20 mm orally. Oral EJPs and anal IJPs were linearly related in amplitude between the two muscle layers. Spatiotemporal maps generated from simultaneous video imaging of the movements of the colon, combined with intracellular recordings, revealed that some LM contractions orally could be correlated in time with IJPs in CM cells anally. N(omega)-nitro-L-arginine (L-NA; 100 microM) abolished the IJP in LM, whereas a prominent L-NA-resistant "fast" IJP was always observed in CM. In summary, in stretched preparations, synchronized EJPs in both LM and CM orally are generated by synchronized firing of many ascending interneurons, which simultaneously activate excitatory motor neurons to both muscle layers. Similarly, synchronized IJPs in both LM and CM anally are generated by synchronized firing of many descending interneurons, which simultaneously activate inhibitory motor neurons to both muscle layers. This synchronized motor activity ensures that both muscles around the entire circumference are excited orally at the same time as inhibited anally, thus producing net aboral propulsion.     

Histamine H3 receptors in the guinea pig ileum: evidence for a postjunctional location.

The effect of the selective histamine H3 receptor agonists (R)alpha-methylhistamine, (R)MHA and immepip (IMM) on intestinal smooth muscle contractility was investigated on isolated cells from the longitudinal muscle of the guinea pig ileum. (R)MHA (10(-13)-10(-8) M) and IMM (10(-13)-10(-8) M) did not significantly modify the basal length of intestinal cells; in contrast both agonists (10(-15)-10(-11) M) prevented the contraction produced by acetylcholine (10(-7) M). The (S)-isomer of alpha-methylhistamine, (S)MHA, was inactive both on basal contractility and on acetylcholine-induced contractions. The relaxant effect of (R)MHA was not modified by famotidine (10(-7) M), but totally prevented by the selective H3 receptor antagonist clobenpropit (10(-8) M), which per se did not modify either basal contractility or the contractile response to acetylcholine. These data indicate that inhibitory histamine H3 receptors are present on smooth muscle cells of the guinea pig ileum and can be activated by very low concentrations of selective agonists. It is not clear, however, whether they can have a functional importance in the regulation of intestinal contractility in an intact system.     

Structure of zonulae occludentes and the permeability of the epithelium to short-chain fatty acids in the proximal and the distal colon of guinea pig

Summary Absorption of short-chain fatty acids has been studied in the proximal and the distal colon of anaesthetized guinea pigs. Segments were perfused with a solution similar in chemical composition to that of normal colonic fluids. In the proximal colon the permeability of the mucosa was similar for acetate, propionate and butyrate. For acetate the permeability was significantly higher in the proximal than in the distal colon, and the reverse was seen for butyrate. In the distal colon the short-chain fatty acids seem to be absorbed mainly in the undissociated form due to their lipid solubility: a paracellular pathway for the dissociated molecules is of no major importance. In the proximal colon, on the other hand, a considerable portion of acetate and propionate disappears in the ionized form. Light microscopy (semithin sections) and electron microscopy (freeze-fracture replicas) showed remarkable morphological differences between the proximal and the distal colon. Leaky spots with only few strands were present in the zonulae occludentes between the epithelial cells at the surface of the proximal colon. In the distal colon the junctions between the cells were more compact, and significantly more strands separated the lumen from the intercellular space. These results suggest that short-chain fatty acids could be absorbed by a paracellular pathway in the proximal colon, and not in the distal colon. In the proximal colon the number of strands of the zonulae occludentes between surface cells and that between cryptal cells was similar. On the contrary, in the distal colon significantly more strands were present between surface cells than between cryptal cells. Morphological and physiological considerations suggest that absorption of short-chain fatty acids in the crypts is negligible.