Ontogenetic development of proenkephalin A and proenkephalin B messenger RNA in fetal pigs

Summary Using solution hybridization analysis and Northern blotting with complementary RNA probes labelled to high specific activity, levels of proenkephalin A and B mRNA were analyzed throughout prenatal development in the hippocampus and striatum of fetal pigs. A differential time course for the appearance of these opioid precursor mRNAs was observed: in hippocampus, both mRNAs increased linearly throughout development with proenkephalin B mRNA increasing faster than proenkephalin A mRNA. In striatum, both mRNAs behaved similarly, increasing to a maximum level around mid-gestation and declining thereafter. The differences might be attributed to differential localization of the two precursor systems in the tissues and might be of functional relevance.Abberviations PENK A, PENK B Proenkephalin A, B - mRNA Messenger Ribonucleic Acid - cRNA Complementary RNA     

Lamellar cells of sensory receptors and perineural cells of nerve endings of pig skin contain cytokeratins

Summary The lamellar cells of the sensory corpuscles of the pig dermis must be considered to be epithelial cells as they contain cytokeratins. The cytokeratins detected are similar to those found in simple epithelia. Moreover, lamellar cells are embedded in an extracellular matrix reminiscent of the basement membrane of epithelium since it contains laminin and collagen IV. The perineural cells surrounding the nerves of pig dermis present the same features.These results suggest that lamellar cells and perineural cells have the same origin. The nature of the lamellar and perineural cells of the rabbit or human dermis is not as clear since cytokeratins were not detected in those cells. These results, together with recent observations on Merkel cells, may indicate that epithelio-neuronal junctions are a general feature of cutaneous sensory receptors.     

Effects of frustration on behaviour and plasma corticosteroid levels in pigs

Frustration was elicited in pigs by not rewarding the operant response of subjects having learnt to press a panel with their snout to get a food reward. When pigs were exposed singly to this situation, they extinguished their operant responding. Plasma corticosteroid levels increased during frustration and the number of responses emitted was correlated negatively with the initial plasma corticosteroid levels. When two pigs were submitted together to the frustration situation, they developed aggressive behaviour accompanied by increases of plasma corticosteroid levels. These experiments demonstrate that pigs are sensitive to the effects of frustration elicited by the omission of an expected reward.     

Postnatal growth of lung parenchyma in the piglet: Morphometry correlated with mechanics

Background: A previous study of piglet lung growth (Mansell et. al. 1989. J. Appl. Physiol., 67:1422–1427) showed transient stiffness to changes in shape and volume immediately after birth. Later, elastic recoil was found to increase as the lung grew in weight and volume. The present study uses morphometry to test possible structural correlates of these two mechanical changes. Methods: Piglet lungs were fixed near full inflation via the airways during the immediate newborn period (6–12 hours, n=3), at 3–5 days (n=6), 25–30 days (n=5), and 80–85 days (n=3). Morphometry comprised arithmetic and harmonic mean thicknesses of alveolar septae and average mean surface curvature. Measurements of curvature and airspace volume were combined to differentiate alveolar expansion from septal proliferation as mechanisms for volumetric growth. Results: The unique mechanical behavior of the newborn lungs was associated with relatively thick alveolar septae. Marked thinning of the septae and resolution of the stiffness to shape and volume change had occurred by 3–5 days. An increase in elastic recoil during the first postnatal month was found to be associated with simple airspace expansion. The second and third months were characterized by septal proliferation and increase in arithmetic mean septal thickness but elastic recoil did not increase further. Harmonic mean septal thickness and airspace volume per gram of lung tissue did not change over the course of the study. Conclusions: 1) A relative stiffness to shape and volume change in freshly newborn piglet lung is associated with relatively thick alveolar septal walls; 2) postnatal development of piglet lung parenchyma involves septal lengthening and thinning followed by septal proliferation; 3) the initial phase of septal lengthening, rather than the later phase of septal proliferation, is associated with increase in parenchymal recoil. © 1995 Wiley-Liss, Inc.     

Microvasculature of regular and irregular areolae of the areola-gland subunit of the porcine placenta: structural and functional aspects

To examine the vasculature of the areola-gland subunit of advanced pig placenta, tissues from ten animals between 43 and 112 days of gestation were prepared for histology and for scanning electron microscopy of vascular corrosion casts from both maternal and fetal sides. Regular areolae, tributary to one gland only, are round with a wide-meshed and smooth subepithelial capillary network on the maternal side, which is similar to the pre-implantative stage and bordered by an abrupt rim towards the interareolar maternal capillary network. On the fetal side, the capillary network follows papillae which protrude into the areolar cavity or converge to form a ring towards the areolar periphery. Irregular areolae, in contrast, have indistinct boundaries and are characterized by two or more gland openings. The maternal capillary network has moderate density and follows the corrugations, whereas the fetal capillary network is basically two-dimensional with some blunt sinusoidal protrusions. Vessel architecture of both areolar types implies facilitated external inflow of blood into the areola on arteriolar as well as on capillary levels, whereas the outflow from the areolar capillaries comprises venules converging into one or two areolar stem veins, and therefore conducts venous blood in a manner different from that of the interareolar region. It is suggested that this arrangement could favour vascular control mechanisms in uterus, placenta and fetus. On the basis of these observations and the discussion, it is suggested that these areolaspecific vessel systems are important for sustaining the characteristic substance transfers in the areola, the secretion, metabolism and absorption, which according to the literature are not the same in the regular as in the irregular areolar type of the porcine areola-gland subunit of the placenta.     

Binding and specificity of major immunoglobulin classes of preformed human anti-pig heart antibodies

Preformed human anti-pig antibodies isolated from perfused pig hearts were used to analyze the binding of various immunoglobulin classes to cultured pig kidney cells. All anti-pig immunoglobulins (i.e., IgG, IgA, and IgM) were localized on the cell surface by the use of an indirect immunofluorescence technique. Anti-pig immunoglobulins also competed for the pig cell surface epitopes with Griffonia simplicifolia lectin (GS-I-B₄), which is specific for -galactosyl residues. This study provides further evidence that preformed human antibodies recognizing -glactosyl-containing epitopes (anti-gal antibodies) could be an important factor in hyperacute rejection of pig organs.     

Histamine H3 receptor-mediated inhibition of noradrenaline release in pig retina discs

Summary Discs of pig retina were preincubated with ³H-noradrenaline, ³H-dopamine or ³H-serotonin and then superfused. Electrical field stimulation increased the outflow of tritium from discs preincubated with ³H-noradrenaline or ³H-dopamine, but no from discs preincubated with ³H-serotonin. The tritium content at the end of superfusion was similar in discs preincubated with ³H-noradrenaline or ³H-dopamine but about tenfold lower in discs preincubated with ³H-serotonin. The tritium content in discs preincubated with ³H-noradrenaline was markedly reduced when desipramine was present during preincubation but was not affected by selective inhibitors of dopamine and serotonin uptake. The tritium content in discs preincubated with 3Hdopamine and ³H-serotonin, in contrast, was reduced or tended to be reduced by a selective dopamine and serotonin uptake inhibitor, respectively.The electrically evoked overflow of tritium from discs preincubated with ³H-noradrenaline was abolished by tetrodotoxin or omission of Ca²⁺. In discs superfused with desipramine, the electrically evoked overflow was enhanced by phentolamine but not affected by histamine. When both desipramine and phentolamine were present in the superfusion medium, histamine inhibited the evoked overflow (pIC₁₅ 6.85). This effect was mimicked by the histamine H₃ receptor agonist R-(–)--methylhistamine as well as by its S-(+)-enantiomer (pIC₁₅ 7.85 and 5.30, respectively) but not by the H₁ receptor agonist 2-(2-thiazolyl)ethylamine and the H₂ receptor agonist dimaprit (each 10 mol/l). The inhibitory effect of histamine was abolished by the H₃ receptor antagonist thioperamide 0.32 mol/l and attenuated by impromidine 3.2 mol/l but not affected by the H₁ receptor antagonist dimetindene 3.2 mol/l and the H₂ receptor antagonist ranitidine 10 mol/l.The results suggest that, in the pig retina, noradrenaline is taken up into, and released from, noradrenergic neurones (most likely vascular postganglionic sympathetic nerve fibres, less probably tissue-specific noradrenergic neurones of the retina) and that noradrenaline release is subject to modulation via H₃ receptors and probably also a-adrenoceptors.Send offprint requests to E. Schlicker at the above address     

Effects of capsaicin,tachykinins, calcitonin gene-related peptide and bradykinin in the pig iris sphincter muscle

Summary Capsaicin-sensitive sensory neurons of the rabbit iris, by releasing tachykinins, exert a major role in the control of pupil motility in response to various noxious stimuli. However, the contribution of sensory innervation to the regulation of iris smooth muscle tone in other mammals species is not known. We have studied the effects produced by electrical field stimulation, capsaicin, substance P, neurokinin A, calcitonin gene-related peptide (CGRP), and bradykinin in the isolated iris sphincter muscle of the pig.Capsaicin (10 M): a) contracted the isolated sphincter muscle and; b) released immunoreactivity for substance P (SP-LI) and CGRP (CGRP-LI) from this preparation. These two effects were no longer observed at the second exposure to the drug. Electrical field stimulation (10 Hz, 60 V, 0.5 ms for 5 s) produced a biphasic contractile response. The rapid component was inhibited by atropine (1 M), while the delayed response was blocked by previous exposure to capsaicin (10 M).Substance P and neurokinin A consistently produced contraction of the pig iris sphincter muscle, substance P being more potent than neurokinin A. CGRP induced a contractile response in more than 50% of the preparations. The tachykinin antagonist [D-Argl, D-Trp^7,9, Leu¹¹-substance P (3 M) blocked: a) the effect of substance P (1 nM); b) the delayed response to electrical field stimulation and; c) reduced by more than 50% response to capsaicin. Bradykinin (10 M) failed to release either SP-LI or CGRP-LI. The contractile response evoked by bradykinin was unaffected by in vitro pretreatment with capsaicin (10 M).The existence in the pig iris of capsaicin-sensitive sensory fibres releasing neuropeptides and thus regulating sphincter muscle tone is proposed. Send offprint requests to Dr. P. Geppetti at the above address     

5-Hydroxytryptamine receptors with a 5-HT6 receptor-like profile stimulating adenylyl cyclase activity in pig caudate membranes

This study deals with the characterization of 5-hydroxytryptamine (5-HT, serotonin) receptors positively linked to adenylyl cyclase in membranes from pig brain caudate. 5-HT and related agonists induced a concentration-dependent stimulation of adenylyl cyclase activity in pig caudate membranes, with the following rank order of potency (mean pEC₅₀ values): 5-HT (7.1) 5-methoxytryptamine (6.9) > 5-carboxamidotryptamine (5.6) > sumatriptan (<5). Maximal stimulation by 5-HT averaged 35 pmol cyclic AMP/min/mg protein over a basal activity of 159 pmol cyclic AMP/min/mg protein. 5-Methoxytryptamine and 5-carboxamidotryptamine had similar efficacies to that of 5-HT, whereas sumatriptan was about half efficacious. Other compounds known as agonists at some 5-HT receptors were weakly potent (mean pEC₅₀ values <5). They include the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), the 5-HT₄ receptor agonist, renzapride and the 5-HT₂ receptor agonist, (1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane) (DOI). In antagonist studies, methiothepin (0.1 and 1 mol/l) shifted the 5-HT curve to the right with no depression of the E_max, yielding pK_B values of 7.4–8.0. Clozapine (1 mol/l) also produced surmountable antagonism of 5-HT-induced effects (pK_B 6.9). Ketanserin (10 mol/l) weakly antagonized 5-HT (pK_B 5.0). The 5-HT₄ receptor antagonists, tropisetron (ICS 205–930) and SDZ 205–557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester), each at 1 mol/l, did not significantly alter the concentration-response curve of 5-HT. The present receptor shares some characteristics of the recently cloned 5-HT₆ receptor (Monsma et al. (1993) Mol Pharmacol 43:320–327): similar pharmacological profile, location (striatum) and ability to stimulate adenylyl cyclase. It may thus represent the functional 5-HT₆ receptor in its natural environment. Correspondence to: P. Schoeffter at the above address