Pleiotropic effects of mitiglinide in type 2 diabetes mellitus.

This study investigated the effects of mitiglinide in 16 patients with type 2 diabetes mellitus treated with 30 mg/day mitiglinide, divided into three doses given just before each meal, for approximately 12 months. A 450 kcal meal tolerance test was performed at baseline and after 3, 6 and 12 months, and levels of plasma glucose and immunoreactive insulin were measured. Various parameters of glucose metabolism and lipid metabolism, urinary albumin and markers of atherosclerosis, coagulation and fibrinolysis were also determined. Mitiglinide showed a rapid stimulatory effect on insulin secretion and reduced the levels of plasma glucose. The free fatty acid level significantly decreased at 60 min after the meal tolerance test. Mitiglinide also significantly lowered glycosylated haemoglobin and raised 1,5-anhydroglucitol after 6 months, and significantly decreased urinary albumin after 12 months. These data indicate that mitiglinide may have beneficial effects not only on glycaemic control but also on lipid metabolism and urinary albumin excretion, and may have a role in the prevention of the vascular complications of diabetes.

[Pacemaker implanted in right inguinal region following removal of infected generator; report of a case].

We treated a 62-year-old male who had previously undergone a mitral valve plasty and aorto-coronary bypass. One year after the operation, he underwent pacemaker implantation for atrial fibrillation. Two months following implantation, the pacemaker generator was exposed due to a methicillin-resistant Staphylococcus aureus (MRSA) infection. We selected a new catheter route from the right saphenous vein, and implanted a generator under the fascia of the external oblique abdominal muscle. Thereafter, the pacemaker is functioning without trouble and there is no evidence of infection.

Ischemic preconditioning upregulates vascular endothelial growth factor mRNA expression and neovascularization via nuclear translocation of protein kinase C epsilon in the rat ischemic myocardium.

Ischemic preconditioning (IP) exerts cardioprotection through protein kinase C (PKC) activation, whereas myocardial ischemia enhances vascular endothelial growth factor (VEGF) mRNA expression. However, the IP effect or the involvement of PKC on the VEGF expression is unknown in myocardial infarction. We investigated whether IP enhances VEGF gene expression and angiogenesis through PKC activation in the in vivo myocardial infarction model. Sprague-Dawley rats were assigned into the following 3 groups: the sham group; the IP group, which underwent 3 cycles of 3 minutes of ischemia and 5 minutes of reperfusion (IP procedure); and the non-IP group. The latter 2 groups were subsequently subjected to left anterior descending coronary artery occlusion. To examine the involvement of PKC, the PKC inhibitor chelerythrine (5 mg/kg) or bisindolylmaleimide (1 mg/kg) was injected intravenously before the IP procedures. PKCepsilon was translocated to the nucleus after 10 minutes of ischemia after the IP procedure but was not translocated in the non-IP and the sham groups. VEGF mRNA expression 3 hours after infarction was significantly higher in the IP group than in the non-IP and the sham groups. Capillary density in the infarction was significantly higher, whereas the infarct size was smaller in the IP group than in the non-IP group at 3 days of infarction. Chelerythrine but not bisindolylmaleimide blocked all of the IP effects on the nuclear translocation of PKCepsilon, enhancement of VEGF mRNA expression and angiogenesis, and infarct size limitation. These results show that IP may enhance VEGF gene expression and angiogenesis through nuclear translocation of PKCepsilon in the infarcted myocardium.

Resistance to endotoxin shock in transgenic mice overexpressing endothelial nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) plays a central role in the pathogenesis of septic shock. However, the role of the NO produced by endothelial NO synthase (eNOS) in septic shock is still unclear. We examined the effect of chronic eNOS overexpression and the role of eNOS-derived NO in lipopolysaccharide (LPS)-induced septic shock using eNOS transgenic (Tg) mice. METHODS AND RESULTS: LPS was intraperitoneally injected into Tg and control mice. No differences existed in the peak plasma nitrate and nitrate levels induced by LPS between the 2 genotypes. In LPS-treated control mice, blood pressure progressively declined and reached 60% of basal levels (from 97+/-3 to 59+/-3 mm Hg) 24 hours after LPS injection. In contrast, the blood pressure of LPS-treated Tg mice fell only 15% from basal levels (from 84+/-4 to 71+/-4 mm Hg) after the first 6 hours and, thereafter, it remained at this level. LPS-induced increases in the expression of the mRNA of both vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 in the lungs were significantly lower in Tg mice than in control mice. LPS-induced pulmonary leukocyte infiltration and increases in lung water content were also significantly attenuated in Tg mice. Histological examination revealed that lung injury after LPS injection was milder in Tg mice. Furthermore, Tg mice exhibited enhanced survival from LPS-induced septic shock compared with control mice. CONCLUSIONS: Chronic eNOS overexpression in the endothelium of mice resulted in resistance to LPS-induced hypotension, lung injury, and death. These effects are associated with the reduced vascular reactivity to NO and the reduced anti-inflammatory effects of NO.

Serotonin-induced hypercontraction through 5-hydroxytryptamine 1B receptors in atherosclerotic rabbit coronary arteries.

BACKGROUND: Augmented vasoconstriction to serotonin (5-hydroxytryptamine [5-HT]) in atherosclerotic vessels plays a crucial role in the development of myocardial ischemia. We investigated mechanisms for serotonin-evoked hypercontraction in atherosclerotic rabbit coronary arteries. METHODS AND RESULTS: Contractile responses to serotonergic agents of endothelium-denuded coronary arteries from control and Watanabe heritable hyperlipidemic rabbits (WHHL) were examined. WHHL coronary arteries exhibited hypercontraction to 5-HT(1)-receptor agonists; the constrictor threshold concentrations and E:D(50) to serotonin, 5-carboxamidotryptamine, and sumatriptan in WHHL were significantly lower, and the E:(max) in WHHL to these agents were increased 55% to 59% above those of the control. Serotonin-evoked contractions in both groups were inhibited by GR127935 (5-HT(1B/1D) antagonist; 0.1 to 1 nmol/L) and pertussis toxin but not by ketanserin (5-HT(2) antagonist; 0.01 to 1 micromol/L), suggesting that the hypercontraction is most likely mediated by 5-HT(1B/1D) receptors through a pertussis toxin-sensitive pathway. Furthermore, simultaneous measurements of [Ca(2+)](i) and isometric tension of fura-2-loaded arteries revealed that the hypercontraction was concomitant with the augmented elevation of [Ca(2+)](i) in the smooth muscle. The 5-HT(1B) mRNA levels in WHHL coronary arteries increased to 2.5-fold over those in control arteries, whereas neither 5-HT(1D) nor 5-HT(2A) mRNA was detected in either group. CONCLUSIONS: Atherosclerotic rabbit coronary arteries exhibited the enhancement in contraction and Ca(2+) mobilization in response to serotonin. The 5-HT(1B) receptor, which is upregulated by atherosclerosis, most likely mediates the augmenting effects of serotonin.

Alterations in cardiac sarcoplasmic reticulum Ca2+ regulatory proteins in the atrial tissue of patients with chronic atrial fibrillation.

OBJECTIVES: Our purpose was to determine whether atrial fibrillation (AF) patients have alterations in sarcoplasmic reticulum (SR) Ca2+ regulatory proteins in the atrial myocardium. BACKGROUND: Clinically, AF is the most frequently encountered arrhythmia. Recent studies indicate that an inability to maintain intracellular Ca2+ homeostasis with a consequent increase in membrane-triggered activity could be the primary initiating factor in some circumstances, and that cytosolic Ca2+ abnormalities are an important mediator of sustained AF. METHODS: We measured the maximum number of [3H]ryanodine binding sites (Bmax) and the expression levels of ryanodine receptor (RyR) mRNA and calcium-adenosine triphosphatase (Ca2+-ATPase) mRNA in atrial myocardial tissue from 13 patients with AF due to mitral valvular disease (MVD) and 9 patients with normal sinus rhythm (NSR). RESULTS: In AF patients, 1) Bmax was significantly lower in each atrium (0.21+/-0.03 pmol/mg [right], 0.16+/-0.04 pmol/mg [left]) than in the right atrium (0.26+/-0.08 pmol/mg) of NSR patients; 2) Bmax was significantly lower in the left atrium than in the right atrium; 3) Bmax in the left atrium was significantly lower at higher levels of pulmonary capillary wedge pressure; 4) the expression level of RyR mRNA was significantly lower in both the left (1.24 x 10(-2)+/-1.28 x 10(-2)) and right (1.70 x 10(-2)+/-1.78 x 10(-2)) atrium than in the right atrium of NSR patients (6.11 x 10(-2)+/-2.79 x 10(-2)); and 5) the expression level of Ca2+-ATPase mRNA was significantly lower in both the left (5.67 x 10(-2)+/-4.01 x 10(-2)) and right (7.71 x 10(-2)+/-3.56 x 10(-2)) atrium than in the right atrium (12.60 x 10(-2)+/-3.92 x 10(-2)) of NSR patients. CONCLUSIONS: These results provide the first direct evidence of abnormalities in the Ca2+ regulatory proteins of the atrial myocardium in chronic AF patients. Conceivably, such abnormalities may be involved in the initiation and/or perpetuation of AF.

Up-regulation of inositol 1,4,5 trisphosphate receptor expression in atrial tissue in patients with chronic atrial fibrillation.

OBJECTIVES: We examined whether patients with atrial fibrillation (AF) have alterations in atrial inositol 1,4,5 trisphosphate receptors (IP3 receptors). BACKGROUND: Abnormal intracellular Ca2+ homeostasis occurs in chronic AF. The intracellular Ca2+ concentration is regulated by ryanodine and IP3 receptors. We recently reported alterations in ryanodine receptors in atrial tissue from patients in chronic AF. METHODS: We analyzed IP3 receptor expression in the right atrial myocardium from 13 patients with mitral valvular disease (MVD) with AF (MVD/AF), five patients with MVD who had normal sinus rhythm (MVD/NSR) and eight control patients with NSR (tissue obtained during coronary artery bypass surgery). Hemodynamic and echocardiographic data were obtained preoperatively, and an immunohistochemical study was performed on atrial tissue. RESULTS: The relative expression level of IP3 receptor protein was significantly greater in MVD/AF (0.75 +/- 0.26) than it was in MVD/NSR (0.42 +/- 0.13, p < 0.01), and both were significantly above control (0.14 +/- 0.08). The relative expression level of IP3 receptor messenger RNA was significantly greater in the MVD/AF group (0.028 +/- 0.008) than it was in the control group (0.015 +/- 0.004, p < 0.01), but patients with MVD/AF did not differ from patients with MVD/NSR (0.020 +/- 0.006). The relative expression levels of IP3 receptor protein and messenger RNA were higher in patients with left atrial dimension > or = 40 mm, pulmonary capillary wedge pressure > or = 10 mm Hg and right atrial pressure > or = 5 mm Hg. Inositol 1,4,5 trisphosphate receptors were over-expressed in the cytosol and at the nuclear envelope of atrial myocytes in MVD. CONCLUSIONS: Since chronic mechanical overload of the atrial myocardium increased IP3 receptor expression, especially in patients with chronic AF, up-regulation of IP3 receptors may be important in modulating intracellular Ca2+ homeostasis and initiating or perpetuating AF.

[Acute penetrating neck trauma presenting with laryngotracheal injury].

Laryngotracheal injuries are serious complications in the case of penatrating neck trauma which may not commonly in Japan. In the last several decades, many authors have discussed method for accurate evaluation and immediate airway management for patient with laryngotracheal injury. But, standardization of the treatment is still controversial about mandatory exploration or selective exploration. We report 4 cases with fresh laryngotracheal injury due to penetrating neck trauma including 3 suicide attempt patients. In these cases, laryngotracheoplasty used by absorbable material was performed within 8 hours after trauma. Two cases of suicide attempt patients underwent tracheostomy at the lower level of the laryngotracheal injury. After these treatment, fiberoptic bronchoscopy was performed to evaluate the airway for 3 cases except 1 who was dead because of hemorrhagic shock on arrival. In 2 cases, the suture filament existed in the lumen of the larynx and trachea, there were no major granulation in the site of repairment and no infection. Three cases were extubated successfully and discharged without major airway problem. Two cases have psychiatric disease such as depression, so we must consider their psychiatric background in the future. In conclusion, penetrating laryngotracheal trauma, we should consider that serious airway injury may be hidden under the superficial small wounds. Also, rapid local wound exploration and laryngotracheoplasty is important for life-saving, and fiberoptic bronchoscopy is effective to prevent early respiratory complications and has value in the evaluation.

Growth hormone preserves cardiac sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors) and enhances cardiac function in cardiomyopathic hamsters.

OBJECTIVE: Growth hormone (GH) improves cardiac function in experimental models of heart failure and human dilated cardiomyopathy. However, the mechanism by which GH increases myocardial contractility is not entirely clear. Our aim was to examine the effects of GH on cardiac function and cardiac sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors, RyR) in the hearts of UM-X7.1 cardiomyopathic hamsters during the development of heart failure. METHODS: Experimental and healthy control hamsters were examined at the age of 20 weeks. Recombinant human GH (2 mg/kg/day, s.c.) or vehicle was then administered for 3 weeks. We examined (i) the in vivo left ventricular (LV) size and LV systolic function using transthoracic echocardiography, (ii) the density (Bmax) and affinity (Kd) of high-affinity [3H] ryanodine binding sites in crude homogenates from normal and cardiomyopathic hamster hearts. RESULTS: Vehicle-treated UM-X7.1 hamsters exhibited significant increases in left ventricular end-diastolic diameter and end-systolic diameter (LVESd), and a significant decrease in LV fractional shortening (FS). GH-treatment attenuated the increase in LVESd and reduced the LV chamber size, and also significantly increased LVFS. Vehicle-treated UM-X7.1 hamsters exhibited a significantly lower Bmax than control hamsters (0.34 +/- 0.04 vs 0.44 +/- 0.06 pmol/mg, p < 0.05), and the treatment with GH in UM-X7.1 hamsters significantly attenuated the reduction of Bmax (0.42 +/- 0.03 pmol/mg vs vehicle-treated group (0.34 +/- 0.04 pmol/mg), p < 0.05). Kd did not differ significantly between the experimental groups. In normal control hamsters, GH treatment with this dose did not significantly enhance LV systolic function or the density of RyRs. There was no significant difference in terms of the connective-tissue volume-fraction, myocyte size and capillary density between the GH- and vehicle-treated groups of UM-X7.1 hamsters. CONCLUSIONS: GH treatment may improve cardiac function by preserving the density of RyRs and enhancing cellular function in cardiomyopathic hamster hearts.

Distribution of primary afferent fibers projecting from hindlimb cutaneous nerves to the medulla oblongata in the cat and rat.

The dorsal column nuclear complex, one of the most important relays for tactile perception, has well been known to be somatotopically organized. Topographical arrangements of terminal sites of individual cutaneous nerves within the dorsal column nuclei, however, have not been examined systematically, although many studies have been done upon primary afferents to the medulla oblongata, including the dorsal column nuclear complex. Thus, in the present study, distribution of primary afferent fibers projecting from the hindlimb cutaneous nerves to the medulla oblongata was examined in the cat and rat by means of the transganglionic transport method with horseradish peroxidase. Cutaneous primary afferent fibers projecting from the hindlimb to the medulla oblongata were distributed mainly in the ipsilateral gracile nucleus. Terminal labeling in the gracile nucleus was seen at all rostrocaudal levels of the nucleus, occasionally including the nuclear part straddling the midline (the median or accessory nucleus). The labeled axon terminals in the gracile nucleus were more densely distributed in the middle and caudal parts of the nucleus than in the rostral part. Although the fields of termination of the hindlimb cutaneous nerves overlapped in the gracile nucleus, the foci of the terminal labeling of the nerves innervating the distal parts of the hindlimb were located more medially or dorsomedially than those of the nerves innervating the proximal parts. Terminal labeling was further found in a small zone immediately medial to the rostromedial border of the external cuneate nucleus. This hitherto undescribed zone (U zone) contained a small cluster of medium-sized neurons in the cat. Although no particular cell cluster was found in the U zone of the rat, convergence of the primary afferent fibers of the cutaneous nerve from the hindlimb appeared to occur as in the U zone of the cat.

Infrahyoid and accessory motoneurons in the Japanese monkey (Macaca fuscata).

The segmental and topographical organization of motoneurons innervating the infrahyoid (IH) and the spinal accessory (AC) muscles was studied in the Japanese monkey (Macaca fuscata) with the retrograde horseradish peroxidase (HRP) method after application of HRP to the peripheral nerve branches supplying the IH and AC muscles. IH motoneurons constitute two distinct slender cell columns, a longer medial and a shorter lateral one. The medial cell column extends from the most caudal level of the hypoglossal nucleus to the lower levels of the second cervical (C2) cord segment. In the medial column, motoneurons supplying the sternohyoid and sternothyroid muscles are distributed at the medullary and C1 levels, while those innervating the omohyoid muscle are primarily distributed at the C2 level. The lateral cell column consists of motoneurons supplying the thyrohyoid muscle and extends from the most caudal level of the hypoglossal nucleus to the middle levels of the C1 cord segment. Axons of thyrohyoid motoneurons follow a dorsomedially directed bent emergent course, making a hairpin turn. AC motoneurons supplying the sternocleidomastoid (SC) and trapezius (TZ) muscles form a single slender cell column extending from the most rostral level of the pyramidal decussation to the middle levels of the C6 cord segment. SC motoneurons are distributed from the most rostral level of the pyramidal decussation to the middle levels of the C3 cord segment, while TZ motoneurons are distributed from the upper levels of the C2 cord segment to the lower levels of the C6 cord segment. At the levels of the C2 and C3 cord segments, both SC and TZ motoneurons are distributed in the AC cell column; the cluster of SC motoneurons is located dorsomedial to that of TZ motoneurons.

Central distribution of efferent and afferent components of the pudendal nerve in macaque monkeys.

Central distribution of efferent and afferent components of the pudendal nerve was studied by the horseradish peroxidase (HRP) method in 13 macaque monkeys, i.e., in nine Japanese monkeys (Macaca fuscata), two rhesus monkeys (Macaca mulatta), and two crab-eating monkeys (Macaca fascicularis). The enzyme was applied to the central cut end of the pudendal nerve; then the monkeys were allowed to survive for 36 to 72 hr. Retrogradely labeled neuronal cell bodies of pudendal motoneurons constituted a slender longitudinal cell column in the ventral horn. The cell column extended from high or middle S1 to high or middle S2 in eight monkeys, from middle or low L7 to high S2 in four monkeys, and from high L7 to middle S1 in a monkey. The cell column appeared to correspond to Onuf's X nucleus in man. No sex difference was recognized in the position of the cell column. The average number of HRP-labeled pudendal motoneurons was larger in male than in female adult Japanese monkeys, whereas no sex difference was found in the average soma diameter of the pudendal motoneurons. Transganglionically labeled axons entered into the spinal cord through the S1 and S2 dorsal roots in 12 monkeys and through the L7 and S1 dorsal roots in one monkey. Labeled axons were distributed ipsilaterally in laminae I-VI and X of the spinal cord at the same and adjacent levels of entry of HRP-labeled dorsal root fibers (from L7 to S3 in 12 monkeys and from L6 to S3 in one monkey).(ABSTRACT TRUNCATED AT 250 WORDS)

Central distribution of afferent and efferent components of the pudendal nerve in cat.

Central distribution of afferent and efferent components of the pudendal nerve was examined in the cat by the HRP method after applying HRP to the central cut end of the pudendal nerve. Retrogradely labeled neuronal cell bodies were located primarily in the feline homologue of the Onuf's X nucleus, constituting a slender longitudinal cell column in the ventral horn of the S1 and S2 cord segments. The Onuf's nucleus was present constantly from middle S1 to high S2 cord segments, and occasionally extended rostrally to high S1 or low L7, and caudally to middle S2, low S2, or high S3 cord segments. No sex differences were observed in the distribution pattern, number, and soma size of labeled neurons in the Onuf's nucleus. Transganglionically labeled dorsal root fibers were found to terminate ipsilaterally in the lamina I of the dorsal horn at levels of lower lumbar, sacral, and higher coccygeal cord segments and the gracile nucleus, and bilaterally with an ipsilateral predominance in the dorsal commissural gray and laminae III, IV, V, and VI of the dorsal horn at levels of lower lumbar, sacral, and higher coccygeal cord segments. Some labeled dorsal root fibers appeared to end ipsilaterally in the regions where the sacral parasympathetic preganglionic neurons have been shown to be located.

Distribution of nociceptin/orphanin FQ precursor protein and receptor in brain and spinal cord: a study using in situ hybridization and X-gal histochemistry in receptor-deficient mice.

Nociceptin/orphanin FQ (N/OFQ) is an opioid-like heptadecapeptide agonist for the opioid receptor homolog, N/OFQ receptor. To explore the precise distribution of the peptide-receptor system, the authors examined the brain and spinal cord from receptor-deficient mice bearing the targeted mutation (morc(m1)), a lacZ insertional mutation in the N/OFQ receptor gene. Precursor protein N/OFQ (preproN/OFQ) mRNA was detected by using in situ hybridization, and the N/OFQ receptor was detected by using X-gal histochemistry. The N/OFQ receptor reflected by lacZ expression was observed at high levels in the dentate gyrus, lateral septum, subparafascicular thalamic nucleus, medial preoptic area, median preoptic nucleus, ventromedial preoptic nucleus, anterior hypothalamic area, paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, auditory brainstem nuclei, pontine dorsal tegmentum, and nucleus of the solitary tract. In situ detection of the N/OFQ receptor mRNA by digoxigenin-labeled riboprobes coupled with tyramide signal amplification in normal and wild-type mice resulted in the regional distribution paralleling the lacZ expression in these regions. PreproN/OFQ mRNA was expressed at high levels in the subparafascicular thalamic nucleus, central gray, central tegmental field, auditory brainstem nuclei, caudal spinal trigeminal nucleus, and spinal dorsal horn. Furthermore, variable levels of expression of the peptide and receptor were seen in distinct sites of the brain and spinal cord. These data indicate a correspondence of the peptide and the receptor in local distribution at limbic, hypothalamic, and brainstem sites. Together with concurrent physiologic and behavioral studies in mutant mice, the results suggest functional roles for the N/OFQ system, including the central regulation of learning and memory, hearing ability, water balance, food intake, and blood pressure.

Vimentin-positive gastric carcinomas with rhabdoid features. A clinicopathologic and immunohistochemical study.

We studied 71 patients with solid-type gastric adenocarcinoma selected from 5,437 surgically resected specimens during the period from 1975 to 1988; six had vimentin-positive adenocarcinomas, and five of these were advanced. One was at an early stage. All six tumors showed the same histologic features and had either a diffuse or alveolar arrangement, with tumor cells having either poor or no cohesiveness. Many tumor cells were round to polygonal, with eosinophilic or clear cytoplasm and large, eccentric vesicular nuclei, as seen in malignant rhabdoid tumors of the kidney. In all cases, the cytoplasm showed coexpression of vimentin and cytokeratin as revealed by double immunostaining. Four of the five cases with advanced carcinoma died of the disease 1 to 6 months after surgery. The cases with vimentin-positive tumors had significantly poorer prognoses than those with vimentin-negative tumors. We also studied adenocarcinomas of various histologic types randomly selected from our file (160 intestinal type and 69 diffuse type of Lauren) but failed to detect any vimentin positive ones. These results indicate that vimentin is expressed in some of the solid-type adenocarcinomas, which have a poor prognosis, and indicating that rhabdoid-like cells may be found in a variety of adenocarcinomas of the stomach.

Production of nerve growth factor by cultured vascular smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto rats.

OBJECTIVE: Nerve growth factor (NGF) is a neurotrophic protein which acts on peripheral sympathetic nerves. Elevated NGF in vascular tissues of young spontaneously hypertensive rats (SHR) has been reported. The aim of the present study was to compare the amount of NGF secreted from cultured vascular smooth muscle cells (VSMC) and mesenteric artery and thoracic aorta segments from SHR and Wistar-Kyoto (WKY) rats. METHODS: VSMC prepared by the enzyme digestion method from the thoracic aortic media of 14-week-old SHR and age-matched WKY rats were subcultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Segments of mesenteric artery and thoracic aorta from 4-week-old SHR and age-matched WKY rats were similarly cultured. The NGF content in conditioned medium was measured using an enzyme immunoassay. The protein content of VSMC was measured by the Lowry method. RESULTS: Total NGF content in the cell culture medium was increased during an exponential growth phase and then gradually decreased during a quiescent phase in both rat strains. There were no significant differences in the levels of NGF secreted from mesenteric artery and thoracic aorta segments between the SHR and WKY rats. The differences in cellular protein content between SHR and WKY rats were very small. CONCLUSIONS: In contrast to the reports of increased NGF in SHR tissues, our data demonstrate that NGF secretion was lower in VSMC from SHR, and was equivalent in mesenteric artery and thoracic aorta segments from SHR and WKY rats. We have no clear explanation for these observations, but the present results indicate that upregulation of NGF in SHR tissues is not responsible for a simple enhancement of NGF synthesis in VSMC, and suggest a breakdown of the regulatory mechanism or mechanisms of NGF gene expression in SHR tissues.

Increased nerve growth factor levels in spontaneously hypertensive rats.

OBJECTIVE: Increased sympathetic innervation has been reported in spontaneously hypertensive rats (SHR); however, the precise mechanisms involved are not yet clear. Nerve growth factor (NGF), a neurotrophic peptide in peripheral sympathetic neurons, is believed to contribute to this phenomenon. METHODS: We measured the content of NGF in SHR and control Wistar-Kyoto (WKY) rats during development. Mesenteric artery, spleen, heart and sciatic nerve were isolated and homogenized. NGF content in the supernatant fractions was measured using a highly sensitive and specific two-site enzyme immunoassay. RESULTS: At 3 weeks of age, SHR had a greater NGF content in the spleen, the sciatic nerve and the mesenteric artery than WKY rats. However, these differences disappeared completely at 12 weeks of age. Cardiac NGF content was slightly lower in 3-week-old SHR and, conversely, higher in 12-week-old SHR than in age-matched WKY rats. CONCLUSIONS: These findings suggest that, except for the heart, the SHR tissues observed overproduce NGF at a young age, leading to enhancement of peripheral sympathetic nervous system activity and the production of vasoconstrictive catecholamines.

Distribution and cerebellar projections of cholinergic and corticotropin-releasing factor-containing neurons in the caudal vestibular nuclear complex and adjacent brainstem structures.

By using immunohistochemistry combined with lesioning and retrograde neuronal labeling techniques, cholinergic neurons and corticotropin-releasing factor-immunoreactive neurons were examined for their distribution, coincidence and cerebellar projections in feline vestibular nuclear complex and adjacent brainstem structures. Cholinergic neurons as revealed here with choline acetyltransferase immunoreactivity were found massively in the abducens and hypoglossal nuclei, dorsal motor nucleus of the vagus nerve and nucleus of Roller; less numerously in the medial vestibular, prepositus hypoglossi and solitary nuclei and the caudal two-thirds of descending vestibular nucleus; and only occasionally in the intercalated and supravestibular nuclei and cell groups f, x and z. Corticotropin-releasing factor-immunoreactive neurons were found clustered in the prepositus hypoglossi nucleus and also in cell groups f and x and the rostral two-thirds of descending vestibular nucleus, less numerously in the medial vestibular, intercalated and solitary nuclei and nucleus of Roller, and only occasionally in the caudal one-third of descending vestibular nucleus, the dorsal motor nucleus of the vagus nerve, supravestibular nucleus and cell group z. The lateral and superior vestibular nuclei did not contain either type of neuron. The two types of immunopositive neurons observed in most of the brainstem nuclei differed in cell size, distribution-pattern and rostrocaudal level of occurrence. While there were many regions which exhibited both types of immunopositive neurons, perikarya colocalizing the cholinergic and peptide markers were not detected in the brainstem. Following unilateral, partial lesioning of the vestibular nuclear complex, corticotropin-releasing factor-immunoreactive mossy fiber terminals (rosettes) disappeared from the ipsilateral flocculus. However, such lesions did not produce clear-cut changes of cholinergic terminals in the vermis. Following retrograde neuronal labeling combined with immunohistochemistry, the two types of immunopositive neurons observed in most of the brainstem sites were found to project to the vermal lobules I-III, IX and X. On comparison of these immunopositive projection neurons with non-immunoreactive, retrogradely labeled neurons, the cholinergic neurons and the peptide-immunoreactive neurons were found to constitute a major part of the total vestibulocerebellar neuronal population. The results indicate chemical heterogeneity in vestibular nuclear complex and cerebellar afferents.

Chronic glucocorticoid administration as well as repeated stress affects the subsequent acute immobilization stress-induced expression of immediate early genes but not that of NGFI-A.

We reported that repeated immobilization for six days attenuates the subsequent acute immobilization stress-induced expression of the immediate early genes c-fos, fos B, jun B and nerve growth factor-induced gene-B (NGFI-B), but not of NGFI-A, in the rat paraventricular hypothalamic nucleus. In this study, we confirmed these findings by means of a time-course study, and further investigated whether the elevated plasma basal glucocorticoid level induced by repeated stress underlies the attenuated response of immediate early genes and the preserved reactivity of NGFI-A. Rats implanted with 100, 200 or 400 mg corticosterone or placebo pellets (control), were immobilized for 1 h and decapitated seven days later. In control rats acute immobilization induced c-fos, fos B, jun B, NGFI-A and NGFI-B messenger RNA in the paraventricular hypothalamic nucleus, whereas all of them except NGFI-A, were significantly reduced in rats given 200 and 400 mg corticosterone implants. The similarity of the results from the two procedures suggests that glucocorticoid is involved in regulating immediate early genes in the paraventricular hypothalamic nucleus under repeated stress and that the NGFI-A gene is not regulated by this mechanism. However, the plasma basal corticosterone level in repeatedly stressed rats was lower than that of rats implanted with 100 mg corticosterone, suggesting that a repetitive stress-induced corticosterone surge also contributes to this mechanism.

Choline acetyltransferase immunoreactivity in the cat cerebellum.

Choline acetyltransferase immunoreactivity was demonstrated in particular projection systems in cat cerebellum by combining immunohistochemistry, retrograde tracing and lesioning paradigms. The monoclonal antibody used in this study recognized a 68,000 mol. wt protein on immunoblots of cat cerebellum and striatum. Choline acetyltransferase immunoreactivity was localized to some neurons and varicose fibers in the cerebellar nuclei, and also to some mossy fibers and endings (rosettes), fiber plexuses around Purkinje cells, granule cells and parallel fibers in the cerebellar cortex. In addition, the presence of choline acetyltransferase-immunoreactive large cells, presumptive Golgi cells, in the granular layer was confirmed. In each cerebellar nucleus, choline acetyltransferase-immunoreactive neurons contained either large, medium-sized or small cell bodies and were distributed evenly in the entire nuclear domain. Large and medium-sized ones were frequently encountered. Choline acetyltransferase-immunoreactive mossy fibers and rosettes were most abundant in the vermal lobules I-III, VIII, IX and the simple lobule, moderately accumulated in the vermal lobules IV-VII, X, crus I and crus II, and less abundant in the paramedian lobule, paraflocculus and flocculus. Some granule cells with prominent dendritic claws and bifurcating parallel axons were immunolabeled in the entire vermis with infrequent occurrence in the remaining cortices. Following unilateral lesioning of the cerebellar nuclei with electrocoagulation or kainate injections, a reduction in number of choline acetyltransferase-immunoreactive fibers occurred ipsilaterally in the cerebellar cortex and contralaterally in the red nucleus, ventrolateral thalamic nucleus and ventroanterior thalamic nucleus. In addition, perikarya of some cerebellothalamic neurons were shown to contain choline acetyltransferase immunoreactivity. The results indicate that some nucleocortical, cerebellorubral and cerebellothalamic projections are cholinergic and that a subpopulation of cholinergic granule cell-parallel fibers exists.