Neuropeptides in the gastrointestinal canal of Necturus maculosus

Summary The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut.Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum.Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa.The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity.It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.     

Embryonic development and molting of the antennal coeloconic no pore- and double-walled wall pore sensilla in Locusta migratoria (Insecta,Orthopteroidea)

Summary The embryonic development of antennal coeloconic sensilla was studied at four stages between 132 and 252 h after oviposition in Locusta migratoria. Initially the anlagen of the sensilla consist of 2–4 sensory cells and 3 enveloping cells. Two additional cells contribute later to the formation of socket and pit. The dendritic outer segments of the sensory cells elongate before the trichogen process grows out (ecdysis type I) with exception of one sensory cell in anlagen of poreless (np) sensilla. Other differences between np and double-walled wall pore (dw wp) sensilla are not visible until at least about 220 h after oviposition. Molting, which was studied in four stages, follows ecdysis type I in both sensillum types. The fourth enveloping cell maintains its tight connection to the socket of the sensillum even after apolysis. Its apical portion is torn off and shed together with the old cuticle. The electron-dense material between the dendritic sheath and the cuticular wall of the peg in np sensilla, which is regarded important for stimulus transmission, is not deposited during retraction of the trichogen cell. The concentric walls and spoke channels characteristic of dw wp sensilla result from deposition of cuticular material around wedge-shaped projections of the trichogen cell. The typical trilaminar 15 nm cuticulin layer is produced only on the ridges of these sensilla. The first cuticular lining of the spoke channels is only 7 nm thick and of a different structure. A flocculent material surrounds the outgrowing trichogen process. It is continuous with the filling of the spoke channels and can thus be considered as component of the stimulus-transmitting material in the functioning intermolt dw wp sensilla.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: I. Receptor Characterisation

Characterisation of receptor-mediated breakdown of inositol phospholipids in rat cortical slices has been performed using a direct assay which involves prelabelling with [3H]inositol. When slices were preincubated with [3H]inositol, lithium was found to greatly amplify the capacity of receptor agonists such as carbachol, noradrenaline, and 5-hydroxytryptamine to increase the amount of radioactivity appearing in the inositol phosphates. Using a large variety of agonists and antagonists it could be shown that cholinergic muscarinic, alpha 1-adrenoceptor, and histamine H1 receptors appear to be linked to inositol phospholipid breakdown in cortex. The large responses produced by receptor agonists allowed a clear discrimination between full and partial agonists as well as quantitative analysis of competitive antagonists for each receptor. Whereas carbachol and acetylcholine (in the presence of a cholinesterase inhibitor) were full agonists, oxotremorine and arecoline were only partial agonists. Very low concentrations of atropine shifted the carbachol dose-response curve to the right and allowed inhibition constants for the antagonist to be easily calculated. The nicotinic antagonist, mecamylamine, was ineffective. Noradrenaline adrenaline were full agonists at alpha 1-adrenoceptors, but phenylephrine and probably methoxamine were partial agonists. Prazosin, but not yohimbine, potently and competitively antagonised the noradrenaline inositol phospholipid response. Mepyramine but not cimetidine competitively antagonised the histamine response. These data provide strong confirmation for the potentiating effect of lithium on neurotransmitter inositol phospholipid breakdown and emphasise the ease with which functional responses at a number of cortical receptors can be characterised.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

CO2 sensitive receptors on labial palps ofRhodogastria moths (Lepidoptera: Arctiidae): physiology,fine structure and central projection

Summary The tips of the labial palps ofRhodogastria possess a pit housing uniform sensilla (Fig. 1), histologically characterized by wall-pores and receptor cells with lamellated outer dendrites (Fig. 2). The receptor cell axons project to glomeruli in the deutocerebrum (cf. Fig. 3) which are not innervated by antennal receptors. From their histology as well as from their central projection these sense organs are identical with palpal pit organs of other Lepidoptera (Lee et al. 1985; Kent et al. 1986; Lee and Altner 1986).Physiologically, the palp-pit receptors respond uniformly; they are most excitable by stimulation with carbon dioxide (Fig. 6) while they exhibit relatively moderate responses to various odorants (Fig. 4). The responses to CO₂ (Fig. 7) show a steep dose-response characteristic. In ambient atmosphere (i.e., ca. 0.03% CO₂) the cells are in an excited condition already; the seeming spontaneous activity exhibited in air is decreased if the preparation is kept under N₂ or O₂ or CO₂-free air (Figs. 7, 10). There is hardly any adaptation of the responses to continuous or repeated stimulation (Fig. 8). Perhaps CO₂ sensitivity is correlated with sensilla characterized by both wall-pores and lamellated dendrites. Pilot tests indicate that CO₂ perception might be widespread in the Lepidoptera (cf. Fig. 12), but the biological significance remains obscure.     

Determination of the region of rDNA involved in polytenization in salivary glands of Drosophila hydei

During the formation of polytene chromosomes in salivary glands of Drosophila hydei, the genes for ribosomal RNA (rDNA) are underreplicated relative to the rest of the genome. We have measured the number of rRNA genes with and without intervening sequences (ivs+ and ivs- genes) in polytene chromosomes of different genotypes. In the group of genotypes having a large number of ivs- rRNA genes polytenization only occurs within the cluster of ivs- genes. In each of these genotypes rDNA polytenization reaches a constant level of 150 ivs- genes per two chromatid sets (2C); X/X constitutions having two nucleolus organizers (NOs) in the diploid set polytenize the same amount of rDNA as X/O constitutions. In the group of genotypes with small ivs- gene numbers, the rDNA region involved in polytenization is longer and has an average length of 1,700 kb per NO, which is constant in these genotypes. Polytenization of rDNA is extended into the cluster of ivs+ genes, in spite of the fact that these genes appear to be nonfunctional. The smaller the number of ivs- genes, the greater the number of ivs+ genes that are polytenized in the NO. In these genotypes, X/X females replicate twice as much rDNA as X/O males, suggesting that both NOs of the diploid set are polytenized. A comparison of the pattern of spacer length heterogeneity in hybrids between different stocks also demonstrates that both NOs are replicated during polytenization.     

Uptake and polar transport of indoleacetic acid in moss rhizoids

In the cucumber ( Cucumis sativus L. cv. Straight Eight) cotyledon expansion assay, cytokinin-stimulated ethylene production was separated from cytokinin-stimulated growth through the use of potassium and calcium salts. Low concentrations of KC1, which dramatically promoted growth induced by cytokinin, inhibited ethylene evolution, while CaCl₂ at a concentration that had no effect on growth, strongly promoted the cytokinin-induced ethylene evolution. In contrast to the growth response, stimulation of ethylene production was not directly related to the presence of potassium or calcium but to their relative concentrations. Concentrations of KCl and CaCl₂ which promoted ethylene evolution singly, strongly inhibited it when mixed together. Low rates of exogenous ethylene had no effect on the growth response. Both the growth and ethylene responses were found to be general cytokinin phenomena. Cotyledon respiration was promoted by KC1, CaCl₂ and cytokinin, but its stimulation was not correlated with either growth or ethylene production. In the presence of KClm cytokinin-induced respiration sharply lowered the content of certain sugars during the large growth response and followed KCl uptake. Analysis of KCl uptake showed that its growth promoting synergism with cytokinin was not due to osmotic effects.     

Biogenic Amine-Stimulated Cyclic Adenosine-3'',5''-Monophosphate Formation in the Rat Carotid Body

The subcutaneous injection of isoprenaline, salbutamol, histamine, and adrenaline to rats, which were subsequently killed by microwave irradiation, resulted in a rapid increase in the cyclic AMP content of the carotid body. On the other hand, noradrenaline, dopamine, adenosine, and 5-hydroxytryptamine, at doses at least 100 times greater than that of isoprenaline, did not significantly alter the cyclic nucleotide content in vivo. The response to isoprenaline was dose related, with an ED50 of 15 micrograms X kg-1, and reached a peak level 1-1.5 min after injection. Incubation of intact carotid bodies with isoprenaline (10(-5) M) in vitro also resulted in a 10-fold increase in cyclic AMP content. The in vivo response to isoprenaline could be blocked stereo-selectively by propranolol, and ICI 118.551, a beta 2-selective antagonist, blocks the isoprenaline-elicited increase in cyclic AMP completely at a dose of 30 micrograms X kg-1; whereas betaxolol, a beta 1-selective antagonist, was ineffective, even at a dose of 300 micrograms X kg-1. Hypoxia (5% oxygen in 95% N2) did not result in a significant increase in the cyclic AMP content, nor did it significantly alter the isoprenaline-stimulated increase in the cyclic AMP content of the rat carotid body. These results suggest that some catecholamines may stimulate cyclic AMP formation by interacting with a beta 2-adrenoceptor in the rat carotid body.     

A chemiluminescent method for measurement of activated oxygen forms in biological fluids and homogenates

A chemiluminescent method for measuring the concentration of activated oxygen species (O₂² and H₂O₂) is described. Its main features are: high sensitivity (10^?9 M H₂O₂), its applicability to systems with high optical absorbance in the visible spectral region, a wide linear dynamic range, and the possibility for recording the kinetics of the processes, in which activated oxygen species are involved.