Low temperature stimulates alpha-melanophore-stimulating hormone secretion and inhibits background adaptation in Xenopus laevis.

It is well-known that alpha-melanophore-stimulating hormone (alpha-MSH) release from the amphibian pars intermedia (PI) depends on the light condition of the animal's background, permitting the animal to adapt the colour of its skin to background light intensity. In the present study, we carried out nine experiments on the effect of low temperature on this skin adaptation process in the toad Xenopus laevis, using the skin melanophore index (MI) bioassay and a radioimmunoassay to measure skin colour adaptation and alpha-MSH secretion, respectively. We show that temperatures below 8 degrees C stimulate alpha-MSH secretion and skin darkening, with a maximum at 5 degrees C, independent of the illumination state of the background. No significant stimulatory effect of low temperature on the MI and alpha-MSH plasma contents was noted when the experiment was repeated with toads from which the neurointermediate lobe (NIL) had been surgically extirpated. This indicates that low temperature stimulates alpha-MSH release from melanotrope cells located in the PI. An in vitro superfusion study with the NIL demonstrated that low temperature does not act directly on the PI. A possible role of the central nervous system in cold-induced alpha-MSH release from the PI was tested by studying the hypothalamic expression of c-Fos (as an indicator for neuronal activity) and the coexistence of c-Fos with the regulators of melanotrope cell activity, neuropeptide Y (NPY) and thyrotrophin-releasing hormone (TRH), using double fluorescence immunocytochemistry. Upon lowering temperature from 22 degrees C to 5 degrees C, in white-adapted animals c-Fos expression decreased in NPY-producing suprachiasmatic-melanotrope-inhibiting neurones (SMIN) in the ventrolateral area of the suprachiasmatic nucleus (SC) but increased in TRH-containing neurones of the magnocellular nucleus. TRH is known to stimulate melanotrope alpha-MSH release. We conclude that temperatures around 5 degrees C inactivate the SMIN in the SC and activate TRH-neurones in the magnocellular nucleus, resulting in enhanced alpha-MSH secretion from the PI, darkening the skin of white-adapted X. laevis.

D2-like dopamine receptor mediates dopaminergic or gamma-aminobutyric acidergic inhibition of melanotropin-releasing hormone release from the pars intermedia in frogs (Rana nigromaculata).

Frogs can adapt to their background by making their skin color lighter or darker as necessary, and this adaptation is regulated by MSH. We investigated the mechanism inhibiting MSH release from the pars intermedia (PI) of the pituitary gland in frogs (Rana nigromaculata) by ultrastructural immunohistochemistry and bioassay using the melanophore index. The PI contained fibers immunoreactive for tyrosine hydroxylase, gamma-aminobutyric acid (GABA), and neuropeptide Y, which made synaptic contacts with MSH cells. The synapses had an asymmetric profile with small round and large-cored synaptic vesicles. The skin of frogs adapted to a white background became darker after administration of 6-hydroxydopamine or autografting of the PI into the anterior chamber of the eye. The skin of autografted frogs became lighter after the administration of dopamine or GABA into the anterior chamber. Lightening of skin color with dopamine was inhibited by a D2 receptor antagonist (sulpiride), and the effect of GABA was blocked by both sulpiride and a GABAA receptor antagonist (bicuculline). These results indicate that MSH release from the PI in frogs may be inhibited by dopaminergic nerves via the D2-like receptor and by GABAergic nerves via the D2-like and GABAA receptors.

Synaptic organization and ultrastructural features of the substance P-receptor-like immunoreactive neurons in the nucleus intermediolateralis of rats.

Synaptic and ultrastructural organization in the intermediolateral nucleus (IML) of rat were investigated with electron microscopy combined with pre-embedding immunohistochemistry for substance P (SP)-receptor (SPr). SPr immunoreactivity in IML was found in the vicinity of the cellular membrane of the perikarya and dendritic profiles of the small sized neurons, ranging from 15 to 25 microns in length. A SPr-immunoreactive (SPr-ir) soma had symmetric or asymmetric synaptic contacts with three to five unlabeled axon terminals. Two different types of axon terminals made synapses on the SPr soma, one contained 20-30 nm pleomorphic vesicles and large dense cored vesicles and the other contained clear pleomorphic vesicles of 30-50 nm in size. Occasionally, SPr-ir dendrites are very closely apposed to the blood capillary. Our present results suggested the possibility that the IML SPr-ir neurons might be activated by several kinds of synaptic inputs and SP provided from blood flow.

Sympathetic and sensory innervation of the rat shoulder joint: a WGA-HRP tracing and CGRP immunohistochemical study.

The innervation of the shoulder joint of the rat was investigated. Nerve origin was assessed by injection of a neuronal tracer (WGA-HRP) into the shoulder joint cavity and calcitonin gene-related peptide (CGRP), which is known to be present in some sensory neurons, was detected immunohistochemically with an anti-CGRP antibody. In the ipsilateral sympathetic and dorsal root ganglia, 133-312 and 12-55 nerve cell bodies were respectively labeled by injection of the tracer. In the sympathetic ganglia, 83% of all labeled cells were found in the stellate ganglion and 17% in the superior cervical ganglion. In the dorsal root ganglia, 75% of the labeled cells were found in C4 and the neighboring ganglia (C4-C5), while the rest were observed in C6-8 and T3. This suggested that the origin of sensory innervation for the shoulder joint was mainly in the mid-cervical cord. CGRP-immunoreactive fibers were found in the synovial capsule of the shoulder joint. These fibers were fine and resembled type 4 axons as classified by Brodal, i.e., nerve related to pain sensation. These findings indicate that sensory nerves from the mid-cervical cord and sympathetic nerves from the cervical ganglion are distributed to the shoulder joint. It is possible that these nerves are related to symptoms such as pain in patients with "frozen" shoulder or other diseases.

Origin of the calcitonin gene-related peptide-immunoreactive nerve fibers in the rat shoulder joint.

The rat shoulder joint capsule is innervated by thin sympathetic and sensory nerve fibers, most of which contain calcitonin gene-related peptide (CGRP). In order to establish the origin and distribution of CGRP-immunoreactive (IR) fibers, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the shoulder joints of rats via a dorsal surgical approach. After WGA-HRP injection, the cervico-thoracic dorsal root ganglia (DRG) were removed and processed using both HRP histochemistry and CGRP immunohistochemistry. In the C4 to C7 DRG, small to medium-sized neurons (20-40 microns) were labeled by this combined method. The number and size of the labeled neurons were measured in the cervical 4th-7th DRG. The number of double-labeled neurons was one quarter of the total number of HRP-labeled neurons and 1/20 of the CGRP-IR neurons. Most of the double-labeled cells were located in the C6 ganglion, and the mean number of double-labeled neurons was 13 at this level. This distribution and function of the CGRP-IR fibers in the rat shoulder joint capsule are discussed.

Quantitative analysis of central terminal projections of visceral and somatic unmyelinated (C) primary afferent fibers in the guinea pig.

In guinea pigs, intracellular labeling of the dorsal root ganglion (DRG) cells with Phaseolus vulgaris-leucoagglutinin (PHA-L) was used to demonstrate the central projections of somatic and visceral afferent C-fibers. The terminations of the afferent fibers were analyzed qualitatively and quantitatively with the aid of camera lucida drawings. Terminal branches of C-fibers of both somatic and visceral origin were, in general, distributed in accord with the organization of the neuropil in lamina of the spinal cord. Terminal boutons arranged from longitudinally coursing fibers were distributed in lamina I, while boutons in lamina II were scattered in an apparent random fashion. The synaptic enlargements were counted in gray matter of the spinal dorsal horn and measured on each terminal branch of a fiber. All synaptic boutons (over one thousand) of somatic fibers were found in the superficial dorsal horn (laminae I and II). More than 60% of the synaptic enlargements of the visceral afferents also were localized superficially (lamina I and adjacent dorsal funiculus) while 10-20% of the visceral enlargements appeared in deeper layers of the spinal cord. Boutons of somatic C-fibers were larger than those of visceral origin. Quantitative data of the unmyelinated afferent fibers are discussed in the context of the sensory functions of myelinated afferent fibers.

Inhibitory Effects of Ellagic Acid on Glucosyltransferases from Mutans Streptococci.

Ellagic acid and its derivatives were found to abrogate the enzymatic activity of the glucosyltransferases (GTases) of mutans streptococci. These compounds preferentially inhibited the water-soluble glucan synthesis by GTase of Streptococcus mutans MT8148 and inhibited the water-insoluble glucan synthesis by GTase of Streptococcus sobrinus 6715. This inhibition resulted in a reduction of cellular adherence of mutans streptococci to a glass surface.

New polyphenolic 5'-nucleotidase inhibitors isolated from the wine grape "Koshu" and their biological effects.

New 5'-nucleotidase inhibitors designated as NPF-88BU-IA, NPF-88BU-IB, NPF-88BU-IIA and NPF-88BU-IIB, respectively, were isolated from the seeds and skin of the wine grape "Koshu". They were purified by solvent extraction, dialysis, and reversed-phase high performance liquid chromatography (HPLC). Their physico-chemical properties revealed these compounds to be polyphenolic substances. The average relative molecular masses of the four were estimated by gel permeation chromatography (GPC) analysis to be 7850, 5950, 11900, and 11300, respectively. They strongly inhibited 5'-nucleotidase activities from snake venom and rat liver membrane, and displayed significant therapeutic activity against Ehrlich ascites carcinoma. They also showed inhibitory effects on the growth of Streptococcus mutans MT8148(c), a primary cariogenic bacterium. Furthermore, these 5'-nucleotidase inhibitors inhibited the glucan formation from sucrose. These results suggest that the 5'-nucleotidase inhibitors can prevent the cause of caries of tooth.

The growth inhibition of Streptococcus mutans by 5'-nucleotidase inhibitors from Areca catechu L.

New 5'-nucleotidase inhibitors named NF-86I, NF-86II were recently isolated from the seeds of Areca catechu L. NF-86I and NF86II showed inhibitory effects on the growth of Streptococcus mutans MT8148(c) and Streptococcus mutans MT6715(g), respectively. In addition, these inhibitors could inhibit insoluble glucan formation from sucrose. NF-86I and NF-86II were found to be polyphenolic substances. Some polyphenols such as tannic acid bind non-specifically to proteins (tannic activity). The 5'-nucleotidase inhibitors that we isolated did not show any such activity. However, the growth inhibitory activity and the inhibitory effect on water-insoluble glucan production were equal to tannic acid. It is therefore considered that these inhibitors bind specifically to the bacterial cell surface. Our findings suggest that the 5'-nucleotidase inhibitors NF-86I and NF-86II may be useful anti-plaque preventing agents.

Phosphatase-inhibitory activity and activation of murine macrophages by new 5'-nucleotidase inhibitors, NPF-86IA, NPF-86IB, NPF-86IIA and NPF-86IIB.

New 5'-nucleotidase-inhibitory polyphenols named NPF-86IA, NPF-86IB, NPF-86IIA and NPF-86IIB were isolated from the seeds of Areca catechu L. The ability of the inhibitors to precipitate gelatin was investigated by microturbidimetry. These inhibitors produced weak turbidity. As 5'-nucleotidase is a kind of phosphatase, we examined the effects of these inhibitors on alkaline and acidic phosphatases. While they showed moderate inhibitory effects on the activity of acidic phosphatases, they did not have any significant effect on the activity of alkaline phosphatase. Therefore, they showed a higher inhibitory effect on the 5'-nucleotidase than the other phosphatases, Murine macrophages were directly stimulated by the 5'-nucleotidase inhibitors.