Descending pathways mediating disynaptic excitation of dorsal neck motoneurones in the cat: facilitatory interactions.

Facilitatory interactions between disynaptic EPSPs evoked from the contralateral tectum, ipsilateral tegmentum and contra- and/or ipsilateral pyramid have been investigated in dorsal neck motoneurones of the cat. Monosynaptic convergence on common intercalated neurones was found from ipsi- and contralateral pyramidal, contralateral tectal and ipsilateral tegmental fibres. In addition, disynaptic facilitation was observed from ipsilateral pyramidal fibres on disynaptic contralateral pyramidal EPSPs. Transection of cortico-fugal fibres in the pyramid showed that the location of the interactions occurred in the lower brain stem, suggesting that reticulospinal neurones are mediating the effects.     

Role of prostaglandin E receptor subtypes in gastroduodenal HCO3- secretion

Gastroduodenal HCO3- secretion is a key process that aids in preventing acid-peptic injury. Endogenous prostaglandins (PGs) play a particularly important role in the local control of this secretion. The secretion of HCO3- in both the stomach and duodenum was increased in response to PGE2 as well as mucosal acidification, the latter occurring with concomitant enhancement of mucosal PG generation. These HCO3- responses in the duodenum were markedly reduced by prior administration of the EP4 antagonist in rats, and profoundly decreased in the animals lacking EP3 receptors but not EP1 receptors. In contrast, gastric HCO3- responses induced by PGE2 and mucosal acidification were prevented by the EP1 antagonist and disappeared in EP1, but not EP3-knockout mice. Consistent with these findings, duodenal HCO3- secretion was stimulated by both EP3 and EP4 agonists but not EP1 or EP2 agonists, while gastric HCO3- secretion was increased by the EP1 agonist but not EP2, EP3 or EP4 agonists. In addition, the HCO3- stimulatory action of sulprostone (EP1/EP3 agonist) in the stomach was inhibited by the Ca2+ antagonist verapamil but not affected by IBMX, the inhibitor of phosphodiesterase, while that in the duodenum was inhibited by verapamil and enhanced by IBMX. Forskolin, the stimulator of adenylate cyclase, increased HCO3- secretion in the duodenum but not the stomach. Thus, the HCO3- stimulatory action of PGE2 in the duodenum is mediated by both EP3 and EP4 receptors being coupled intracellularly with both Ca2+ and cAMP, while that in the stomach is mediated by EP1 receptors, coupled with Ca2+.     

Tetracycline administration restores osteoblast structure and function during experimental diabetes.

Osteopenia is a recognized complication of diabetes mellitus in humans and experimental animals. We recently found that tetracyclines prevent osteopenia in the streptozotocin-induced diabetic rat and that this effect was associated with a restoration of defective osteoblast morphology (Golub et al., 1990). The present study extends these initial ultrastructural observations by assessing osteoblast function in the untreated and tetracycline-treated diabetic rats. After a 3-week protocol, non-diabetic control and diabetic rats, including those orally administered a tetracycline, minocycline (MC), or a non-antimicrobial tetracycline analog (CMT), were perfusion-fixed with an aldehyde mixture; the humeri were dissected and processed for ultracytochemical localization of alkaline phosphatase (ALPase) and Ca-ATPase activities. Some rats from each experimental group received an intravenous injection of 3H-proline as a radioprecursor of procollagen, and the humeri were processed for light microscopic autoradiography. In addition, the osteoid volume in each experimental group was quantitatively examined by morphometric analysis of electron micrographs. During the diabetic state, active cuboidal osteoblasts in the endosteum of control rats were replaced by flattened bone-lining cells that contained few cytoplasmic organelles for protein synthesis (Golgi-RER system), and active transport (mitochondria). Treating diabetic rats with MC, and even more so with CMT, appeared to "restore" osteoblast structure. During diabetes, bone-lining cells incorporated little 3H-proline or secreted little labeled protein and produced only a very thin osteoid layer. Tetracycline administration to the diabetics increased both the incorporation of 3H-proline by osteoblasts and their secretion of labeled protein toward the osteoid matrix, in a pattern similar to that seen in the non-diabetic controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caveolar and intercellular channels provide major transport pathways of macromolecules across vascular endothelial cells

Serum macromolecules are transported through the vascular endothelial layer to the interstitium via the caveolae and interendothelial clefts, but the nature of the permeability of these structures is unknown, and the manner of caveola-vesicle transport is controversial. We have developed a method of detecting macromolecular channels using an in situ HRP perfusion into arteries previously perfused with aldehyde and random conventional sectioning for electron microscopy. Using unbiased morphometry, 4.75% of the abluminal caveolae and 15.13% of the intercellular clefts were the tracer-positive in rat aortic endothelium. In rat aortas treated with N-ethylmaleimide, all caveolae and most free vesicles in the cytoplasm except those around the Golgi area were HRP-positive in the endothelial cells; 1.48% of abluminal caveolae were structurally recognized as caveolar channels through the endothelial layer in a plane of single section. The length density of the abluminal caveolae was decreased to about 80% to the physiological control level whereas the larger invaginations were more frequently observed. Moreover 96.17% of the intercellular clefts were HRP-positive. We suggest that a flexible channel-system functions extensively as a macromolecular transport pathway in the arterial endothelium in vivo because the tracer-labeled abluminal caveolae and intercellular clefts should be opened to the luminal surfaces methodologically. We therefore propose that caveolar channels, rather than transcytosis, provide a mechanism of caveola-vesicle transport in the endothelial cells, because free vesicles involved in transcytosis were few in number.     

Conjugation of DNA fragments to protein carriers by glutaraldehyde: immunogenicity of oligonucleotide-hemocyanin conjugates

The practical realization of the concept of specific immunotherapy for systemic lupus erythematosus (SLE) has been hampered, thus far, by an inability to link DNA fragments to carrier protein. In this paper, a novel technique is described, in which glutaraldehyde is the linking agent. A 2-stage method was used to link oligonucleotides to a soluble protein carrier, such as keyhole limpet hemocyanin (KLH) or human gamma globulin (HGG), whereas a 1-stage technique was sufficient to link oligonucleotides to sheep red cells. Both the ultraviolet absorbance spectrum and diphenylamine assay demonstrated that oligonucleotides were coupled to soluble protein. The conjugate of oligonucleotide to protein carrier appears to be recognized by anti-DNA antibody since oligonucleotide linked to either KLH or HGG inhibited the binding of anti-DNA antibody in vitro, and oligonucleotide-coupled sheep cells are agglutinating by seropositve sera from lupus patients. In addition, oligonucleotide-KLH raised hemagglutinating antibody to denatured DNA in C57BL/6, DBA/2 or NZB mice, as well as IgG antibody as detected by SPRIA in C57BL/6 and DBA/2 mice. The significance of this new method for the development of an antigen specific therapy of SLE is discussed.     

gamma-Aminobutyric acid-induced response in rat dissociated paratracheal ganglion cells.

1. The pharmacologic properties of gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) were studied in the paratracheal ganglion cells freshly dissociated from 7- to 10-day-old rat trachea in a whole-cell recording mode by the use of a conventional patch-clamp technique. 2. GABA- and muscimol-induced currents increased sigmoidally in a concentration-dependent manner, and both currents reversed at approximately -3 mV, which was close to the Cl- equilibrium potential (ECl). 3. Strychnine (STR) at low concentration and bicuculline (BIC) inhibited GABA response competitively, whereas STR at the higher concentrations, benzylpenicillin (PCG), or picrotoxin (PTX) inhibited noncompetitively. Inhibition of GABA response by PCG but not other antagonists was voltage dependent, indicating that PCG acts as a Cl- channel blocker. 4. The concentration-response curve of pentobarbital sodium (PB)-induced ICl was bell shaped. At concentrations higher than 10(-3) M, both the peak and plateau currents decreased, and a transient "hump" current appeared immediately after washing out PB. In the presence of PB, the concentration-response curve of GABA shifted toward left without changing the maximum response. 5. Although diazepam (DZP) at concentration used did not induce a response, it potentiated the GABA response in a concentration-dependent manner between 10(-8) and 10(-6) M. DZP also caused a parallel shift toward left in the concentration-response curve of GABA. 6. PB or DZP further enhanced the GABA response in the presence of the other agent. 7. It is concluded that the properties of GABAA receptors in the paratracheal ganglion cells are essentially similar to those reported in other preparations.     

Effects of exhaustive exercise on biochemical characteristics of sarcoplasmic reticulum from rat soleus muscle

This study examined the effects of acute high-intensity and moderate-intensity exercise on Ca2+-stimulated adenosine triphosphatase (ATPase) activity and the Ca2+ and ATP dependence of Ca2+-ATPase of the sarcoplasmic reticulum (SR) in the soleus muscle. The rats were run on 10% grade at 50 m min(-1) or 25 m min(-1) until fatigued (avg. time to exhaustion 2.8 and 87.7 min, respectively). The catalytic activities of SR Ca2+-ATPase were significantly depressed immediately after both types of exercise. Kinetic analyses demonstrated that the Ca2+ affinity of Ca2+-ATPase was elevated by both types of exercise adopted in the present investigation whereas the increase in the ATP affinity was brought about by only high-intensity exercise. These results suggest that exhaustive exercise may induce in slow-twitch muscle fibre the environmental changes, which adversely affect SR Ca2+-ATPase activity and can overcome the positive influence arising from the increase in the Ca2+ and/or ATP affinities of SR Ca2+-ATPase.     

Autoradiographic studies using L-[(14)C]DOPA and L-DOPA reveal regional Na(+)-dependent uptake of the neurotransmitter candidate L-DOPA in the CNS

We previously proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter in the CNS. Receptor and transporter molecules for L-DOPA, however, have not been determined. In the present study, in order to localize the uptake sites of L-DOPA in the CNS, we performed autoradiographic uptake studies using L-[14C]DOPA and L-[3H]DOPA in the uptake study on rat brain slice preparations, and further analyzed the properties of L-DOPA uptake. Image analysis of the L-[14C]DOPA autoradiogram showed a unique heterogeneous distribution of uptake sites in the brain. The intensity was relatively high in the cerebral cortex, the hypothalamus, the cerebellum and the hippocampus, while the density was moderate or even low in the striatum and the substantia nigra. L-DOPA and phenylalanine, but not dopamine (10mM) were able to almost completely inhibit the uptake of L-[14C]DOPA to basal levels. Microautoradiographic studies using L-[3H]DOPA revealed accumulation of dense grains in the median eminence, the supraoptic nucleus of the hypothalamus, the cerebral cortex (layer I) and the hippocampus. In the cerebellum, grains formed in clusters surrounding the Purkinje cells. This grain accumulation was concluded to be in Bergmann glial cells, since the morphological pattern of grain accumulation was similar to that of the immunoreactivity of the glutamate aspartate transporter, a marker protein for Bergmann glial cells. In the hippocampus, the grain density significantly decreased under Na(+)-free conditions. In addition, grain density also decreased in the absence of Cl(-). In contrast, grains in the choroid plexus and the ependymal cell layer, were not affected by the absence of Na(+). These findings indicated that the uptake of L-DOPA occurs via various types of large neutral amino acid transport mechanisms. It appears that neuronal and/or glial cells, which take up L-DOPA in a Na(+)-dependent manner, exist in the CNS. Our finding further supports the concept that L-DOPA itself may act as a neurotransmitter or neuromodulator.