First recorded outbreak of yellow fever in Kenya, 1992-1993. II. Entomologic investigations

OBJECTIVES: The physiology of the female sexual response and its molecular mediators remain poorly understood. Nitric oxide (NO) is synthesized in neurons and is a potent relaxor of vascular and nonvascular smooth muscle. In this study, we hypothesize that vaginal atrophy and declining sexual function during menopause may be NO dependent. Using the rat as an experimental model, we examined the expression and topologic localization of vaginal NO synthase (NOS) and the concomitant induction of apoptosis under normal and estrogen-depleted conditions. METHODS: Thirty rats were categorized into six groups on the basis of phase of the estrous cycle or estrogen status after oophorectomy. The expression and cellular localization of NOS was examined in frozen sections using specific antibodies against neuronal (N-NOS) and endothelial NOS (E-NOS). Apoptotic cells were identified in situ using the terminal transferase technique (TUNEL). Trichome staining was performed in all specimens to determine smooth muscle/collagen ratios. RESULTS: N-NOS immunoreactivity was localized to nerve fibers supplying vaginal smooth muscle, perivascular nerve plexuses, and lamina propria. E-NOS was localized to vascular endothelium and perivascular smooth muscle fibers. Both E-NOS and N-NOS expression in intact cycling animals was highest during proestrous and lowest during metestrous. After oophorectomy, levels of both N-NOS and E-NOS declined substantially compared with those of intact animals, and there was a parallel induction of apoptosis. Estrogen withdrawal also resulted in increased vaginal atrophy, intramural collagen accumulation, and perivascular wall thickening, as identified by trichome staining. Estrogen replacement resulted in a significant increase in E-NOS and N-NOS expression, as well as diminished apoptosis and vaginal atrophy. CONCLUSIONS: This cellular distribution of NOS in the rat vagina suggests that NO may modulate both vaginal blood supply and vaginal smooth musculature. Estrogen appears to play a critical role in concomitantly regulating vaginal NOS expression and apoptosis in nerves, smooth muscle, vascular endothelium, and epithelium of the rat vagina. These findings may have significant clinical implications for the pathophysiology of postmenopausal female sexual dysfunction.     

Colocalization of nitric oxide synthase and some neurotransmitters in the intramural ganglia of the guinea pig urinary bladder

The distribution of nitrergic neurons was investigated by using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry in wholemount preparations of the urinary bladder in guinea pigs. Both NADPH-d+ and NOS+ neurons were located predominantly in the bladder base. Double staining showed that 70.9% of the NADPH-d+ neurons coexpressed NOS. Acetylcholinesterase histochemistry revealed that a majority of the intramural neurons were reactive, and about half of them (51.4%) were double labelled for NOS. Tyrosine hydroxylase-positive neurons were also distributed mainly in the bladder base but in a neuronal population that was separate from the preponderant NADPH-d+ neurons. Vasoactive intestinal polypeptide immunoreactivity was also detected in the some of intramural ganglion cells, in which 21.3% of them coexpressed NADPH-d. Calcitonin gene-related peptide and substance P immunoreactivities were confined to nerve fibers, often in close association with NADPH-d+ cells or extended along the blood vessels. These results have demonstrated the colocalization of NADPH-d and NOS in the majority of intramural ganglion cells. Many of the nitrergic neurons are apparently cholinergic, indicating that they are parasympathetic postganglionic neurons, and this underscores NO as the major neuromodulator in the parasympathetic nerves in the bladder walls. The localization of vasoactive intestinal polypeptide in nitrergic neurons suggests that the peptide may complement NO for regulation of micturition reflex. The close relationship of NADPH-d-reactive intramural neurons with calcitonin gene-related peptide and substance P fibers, most probably derived from dorsal root ganglion cells, suggests that NO released from the local neurons may exert its influence on the sensory neural pathways in the urinary bladder.     

NADPH diaphorase activity in mammalian retinas is modulated by the state of visual adaptation

NADPH diaphorase histochemistry is commonly used to identify cells containing nitric oxide synthase (NOS), the enzyme catalyzing the production of nitric oxide from L-arginine. NADPH diaphorase activity and NOS immunostaining was demonstrated in different cells of the vertebrate retina; photoreceptors, horizontal cells, amacrine cells, ganglion cells, and Müller cells. However, the physiological role of nitric oxide (NO) in the retina has yet to be elucidated. In this study, we tested the assumption that NADPH diaphorase activity in the retinas of rabbits and rats depended on the state of visual adaptation. In the rabbit, light adaptation enhanced NADPH diaphorase activity in amacrine cells and practically eliminated it in horizontal cells. Dark adaptation induced the opposite effects; the NADPH diaphorase activity was reduced in amacrine cells and enhanced in horizontal cells. Retinas from eyes that were injected intravitreally with L-glutamate exhibited a pattern of NADPH diaphorase activity that was similar to that seen in dark-adapted retinas. In rats, the NADPH diaphorase activity of amacrine and horizontal cells exhibited adaptation dependency similar to that of the rabbit retina. But, the most pronounced effect of dark adaptation in the rat's retina was an enhancement of NADPH diaphorase activity in Müller cells, especially of the endfoot region. Assuming that NADPH diaphorase activity is a marker for NOS, these findings suggest that NO production in the mammalian retina is modulated by the level of ambient illumination and support the notion that NO plays a physiological role in the retina.     

Modulation of quinolinic acid-induced depletion of striatal NADPH diaphorase and enkephalinergic neurons by inhibition of nitric oxide synthase

The present study was designed to examine the role of nitric oxide (NO) in quinolinic acid (QUIN)-induced depletion of rat striatal nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase and enkephalinergic neurons. Intrastriatal injection of QUIN produced a dose-dependent decrease in NADPH diaphorase and enkephalin positive cells, with cell loss being evident following the injection of 6 and 18 nmol QUIN, respectively. To evaluate the role of NO in QUIN-induced toxicity, animals were pretreated with the non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine (l-NAME) or the selective neuronal NOS inhibitor, 7-nitro indazole (7-NI). l-NAME (2x250 mg/kg, i.p. 8 h apart) maximally inhibited striatal NOS activity by 85%, while 7-NI (50 mg/kg, i.p.) maximally inhibited striatal NOS activity by 60%. Pretreatment with l-NAME or 7-NI potentiated the loss of NADPH diaphorase neurons resulting from intrastriatal injection of low doses of QUIN (18 nmol). Neither NOS inhibitor had any effect on the loss of striatal NADPH diaphorase neurons induced by a higher dose of QUIN (24 nmol). In contrast, 7-NI partially prevented the QUIN (18 and 24 nmol)-induced loss of enkephalinergic neurons, while l-NAME had no effect. These results indicate that NO formation may play a role in QUIN-induced loss of enkephalinergic neurons, but not in the loss of NADPH diaphorase neurons.     

Innervation of the striated muscle of the membranous urethra of the male dog

PURPOSE: To identify the functional innervation of the striated muscle layer of the post-prostatic urethra of male dogs. MATERIALS AND METHODS: Detailed anatomic dissection of the pelvic and pudendal nerves was carried out. The pressure and contractile responses to stimulation of these nerves were recorded in vivo and in vitro. RESULTS: Small branches of the pelvic nerve entered the membranous urethra but passed through the striated muscle to the inner smooth muscle layer. Stimulation of the nerve with 1 msec pulses at 10 Hz produced a slow contraction of the urethra which was unaffected by d-tubocurarine. Pudendal nerve branches entered the striated layer from the caudal end. Stimulation produced a rapid, visible contraction that was abolished by d-tubocurarine. Field stimulation of isolated strips of striated muscle resulted only in rapid, d-tubocurarine sensitive contractions. CONCLUSIONS: The striated muscle of the membranous urethra is innervated exclusively by the pudendal nerve.     

Neurotransmitters in the human urethral sphincter in the absence of voiding dysfunction

The purpose of this study was to elucidate the neuroregulation of sphincteric relaxation by investigating the density of nerves containing acetylcholine, noradrenaline, neuropeptide Y (NPY), galanin, vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) in the urethral sphincter in patients without a voiding disorder. The complete urethral sphincter (from the bladder neck to beyond the striated external sphincter) was excised from four male and four female adult cadavers and one male and one female fetus. In transverse paraffin or cryostat sections, the above transmitters were identified by histochemical methods. The striated sphincter was densely innervated by cholinergic nerves. Adrenergic nerves next to striated fibers were rare, but were present in all patients. NPY was seen rarely along striated fibers. In the smooth sphincteric component, noradrenaline-, acetylcholine-, NPY- and galanin-reactive nerves were observed frequently. Only functional studies can clarify the clinical implications of these results. Judging from NPY's scarcity in the striated sphincter no efferent function is anticipated. In the smooth component the frequent appearance of NPY, galanin and noradrenaline suggests a regulatory role for these transmitters.     

Inducible NO synthase II and neuronal NO synthase I are constitutively expressed in different structures of guinea pig skeletal muscle: implications for contractile function

The expression of NOS isoforms was studied in guinea pig skeletal muscle at the mRNA and protein level, and the effect of NO on contractile response was examined. Ribonuclease protection analyses demonstrated NOS I and NOS II mRNAs in diaphragm and gastrocnemius muscle. In Western blots, NOS I and NOS II immunoreactivities were found in the particulate but not the soluble fraction of skeletal muscle. NOS activity was found almost exclusively in the particulate fraction. About 50% of this activity was Ca2+ independent. In immunohistochemistry, the anti-NOS I antibody stained distinct membrane regions of muscle fibers. The most intense staining was seen in neuromuscular endplates identified by labeling with alpha-bungarotoxin. The anti-NOS II antibody labeled muscle fibers that contained alkali-labile myosin ATPase (type I fibers). NOS II was located to intracellular structures and was also seen in "specific pathogen-free" animals. Pretreatment of guinea pigs with bacterial lipopolysaccharide (LPS) markedly intensified NOS II staining. Significant NOS III immunoreactivity was detected only in vascular endothelium. In functional experiments, tetanic muscle contractions were induced in diaphragm and gastrocnemius muscle by electrical stimulation of the innervating nerves. Pretreatment of guinea pigs with LPS or addition of S-nitroso-N-acetyl-D,L-penicillamine to the organ bath markedly decreased tetanic contractions. N(G)-nitro-L-arginine, on the other hand, increased contractile force and reversed the effect of LPS. Our data indicate that NOS II and NOS I are expressed in different structures of skeletal muscle and are involved in the regulation of contractile response.     

Effect of 7-nitro indazole on quinolinic acid-induced striatal toxicity in the rat

Nitric oxide (NO) is implicated as a mediator of cell death in models of neurodegenerative disease. However, the precise role of NO in neuronal degeneration remains controversial. In the present study we employed 7-nitro indazole (7-NI), reportedly a selective inhibitor of neuronal nitric oxide synthase (nNOS) in vivo, to investigate the possible involvement of NO in quinolinic acid (QA)-induced striatal toxicity in the rat. Intrastriatal injection of QA (30 nmol) caused loss of NADPH diaphorase (48%), NOS (48%) and acetylcholinesterase (AChE; 22%) positive neurones and a loss of NOS activity (78%) in striatal homogenates. 7-NI (30 mg kg-1, i.p. every 4 h for 20 h) did not affect the loss of NADPH diaphorase (52%), NOS (52%) and AChE (16%) positive neurones or the loss of NOS activity (66%) in striatal homogenates. The present study does not support a role for NO in QA-induced striatal toxicity.     

Distribution of nitric oxide synthase in the esophagus of the cat and monkey

The distribution of nitrergic neurons and processes in the esophagus of the cat and monkey was studied by light microscopic immunocytochemistry using a specific antibody against purified rat brain nitric oxide synthase and immunoperoxidase procedures. Immunoreactive nerve fibers were found pervading the myenteric plexus, submucous plexus and plexus of the muscularis mucosae, and particularly in the lower esophagus a few immunoreactive fibers entered the epithelium as free nerve endings, some of which derived from perivascular fibers. In the upper esophagus immunoreactive motor end-plates were found in the striated muscle. Thirty-forty-five percent of neuronal cell bodies found in the intramural ganglia and along the course of nerve fiber bundles were immunoreactive and were of the three morphological types earlier described. In the intramural ganglia immunoreactive nerve fibers formed a plexus in which varicose nerve terminals were in close relation to immunoreactive and non-immunoreactive neurons. The intramural blood vessels that crossed the different layers of the esophageal wall were surrounded by paravascular and perivascular plexuses containing immunoreactive nerve fibers. The anatomical findings suggest that nitric oxide is involved in neural communication and in the control of peristalsis and vascular tone in the esophagus. In the lower esophagus a few nitrergic nerve fibers are anatomically disposed to subserve a sensory-motor function.     

Decreased sensitivity in the membranous urethra after orthotopic ileal bladder substitute

PURPOSE: The effect of cystoprostatectomy with orthotopic substitution on membranous urethral sensation and subsequent urinary continence is unknown. We determined the sensory threshold for electrical stimulation of the membranous urethra and correlated it with continence, nerve sparing surgical technique and potency. MATERIALS AND METHODS: The sensory threshold was measured in a control group of 35 men before radical prostatectomy or cystoprostatectomy and in 47 men after cystoprostatectomy and ileal bladder substitution. RESULTS: The sensory threshold of the membranous urethra was 9+/-2 in the control group compared to 27+/-11 mA. in the postoperative group (p<0.001). Patients with daytime continence had a threshold of 24+/-9 compared to 39+/-10 mA. in incontinent patients (p<0.001). We were unable to show any correlation between the sensory threshold in patients with (25+/-10 mA.) and without (31+/-11 mA.) attempted nerve sparing surgery (p = 0.1) nor between potent (25+/-12 mA.) and impotent (27+/-11 mA.) patients (p = 0.4). CONCLUSIONS: Sensitivity in the membranous urethra decreased in patients after cystoprostatectomy and ileal bladder substitution. Urethral sensitivity in the sphincter area was better in continent than incontinent patients. Since we were unable to find any correlation between the sensory threshold and nerve sparing surgery or potency, it may be assumed that at least part of the sensory fibers to the membranous urethra pass through the pudendal nerve and/or the intrapelvic extrapudendal nerve fibers.     

The superior gluteal artery perforator flap

The neuronal isoform of nitric oxide synthase (nNOS, termed also NOS-I) is expressed in normal adult skeletal muscle, suggesting important functions for NO in muscle biology. However, the expression and subcellular localization of NOS in muscle development and myoblast differentiation are largely unknown. In the present study, NOS was immunolocalized with isoform-specific antibodies in developing muscle and in differentiated myoblast cultures (mouse C2C12) together with histochemical NADPH-dependent diaphorase activity that is blocked by specific NOS inhibitors and therefore designated as NOS-associated diaphorase activity (NOSaD). Western blot analysis revealed immunoreactive bands for NOS-I-III in lysates from perinatal and adult muscle tissue and C2C12-myotubes that comigrated with prototypical proteins. In embryonic skeletal muscle, but not in adult myofibers, diffuse cytosolic staining and lack of sarcolemmal NOSaD activity and NOS-I immunoreaction were evident. In both myoblasts and fusioned myotubes, NOSaD and NOS isoforms I-III colocalize in the cytosol. Additionally, members of the sarcolemmal dystrophin-glycoprotein complex (i.e., dystrophin, adhalin, beta1-dystroglycan) immunolocalize in the cytosol of differentiating myoblasts, whereas anti-dystrophin and anti-beta1-dystroglycan clearly delineate the sarcolemma in myotubes. Thus, expression of NOS isoforms I-III and NOSaD is cytosolic in fusion-competent myoblasts during myotube formation in vitro. Interaction of NOSaD/NOS-I with the sarcolemmal dystrophin-complex known from mature myofibers is apparently lacking in prenatal muscle development and differentiating myoblasts. Localization of NOS isoforms thus characterized in myogenic cultures may help further to investigate regulated NO formation in muscle cells in vitro.     

Intercomparison of DNA sizing ladders in electrophoretic separation matrices and their potential for accurate typing of the D1S8O locus

The expression of nitric oxide synthase (NOS) in the mucosa of the canine colon was investigated with in situ hybridzation, immunohistochemistry (using isoform specific antibodies), western analysis, and NADPH diaphorase (NADPH-d) histochemistry. In situ hybridization using a common probe for known isoforms of NOS showed that NOS mRNA was strongly expressed in mucosal cells. A gradient in the degree of hybridization was noted from the base of the crypts to the luminal surface. This gradient was also apparent using an endothelial NOS (eNOS)-specific probe. Neural NOS-like immunoreactivity (nNOS-LI) was observed in columnar epithelial cells, and the same population of cells was stained with NADPH-d. Endothelial NOS-like immunoreactivity (eNOS-LI) was also found in mucosal cells; however, this eNOS-LI was confined to mucous cells. These cells were not stained with NADPH-d. The existence of eNOS in mucosal cells was confirmed by in situ hybridization using the probe which specifically hybridized with mRNA of eNOS and by western blots which demonstrated the expression of a 135-kDa protein in mucosal homogenates. The differential expression of NOS isoforms and the gradient in expression along the length of the crypts suggest complex roles for NO in the development of colonic epithelial cells and in secretion and transport functions of the colonic mucosa.     

Growth hormone enhances regeneration of nitric oxide synthase-containing penile nerves after cavernous nerve neurotomy in rats

PURPOSE: As growth hormone has been reported to improve nerve regeneration, we studied the effect of rat growth hormone (GH) on the regeneration of nitric oxide synthase (NOS)-containing penile nerves and the neurons in the pelvic ganglia after unilateral cavernous nerve neurotomy in rats. MATERIALS AND METHODS: Male rats were divided into three groups: sham operation (n = 14); unilateral neurotomy of a 5 mm. segment of the cavernous nerve (n = 14) with subsequent injection of buffer solution only; and unilateral neurotomy with GH injection (n = 14). Electrostimulation of the intact cavernous nerve was performed at 1 and 3 months. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining was used to identify NOS in penile nerve fibers of the mid-shaft segment and in neurons of the pelvic ganglia. RESULTS: One month after unilateral neurotomy, both the buffer alone and GH-treated groups showed a significant decrease in NOS-containing nerve fibers in the dorsal and intracavernosal nerves on the side of neurotomy. At 3 months, the number of NOS-containing nerve fibers in the buffer alone group did not increase, while the GH-treated group showed a significant increase. In the GH-treated group at 3 months, more NOS-positive neurons in the pelvic ganglia were found on the intact side than on the side of neurotomy (p <0.034), indicating that the regeneration derives from pelvic ganglion neurons on the intact side. Furthermore, electrostimulation in the GH-treated group revealed a greater maximal intracavernosal pressure and a shorter latency period at 3 months than in those given buffer alone. CONCLUSIONS: Our results show that GH injection significantly enhances the regeneration of NOS-containing fibers in the dorsal and intracavernosal nerves after unilateral cavernous nerve injury. We believe that GH administration may present a new and more physiologic approach to the treatment of erectile dysfunction after radical pelvic surgery.     

The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide

OBJECTIVES: The external striated urethral sphincter (rhabdosphincter) is a tubular muscle sleeve that extends from the prostato-membranous urethra and perineal membrane to the bladder neck. The male rhabdosphincter neuroanatomy remains unclear, and a better understanding of its innervation may provide insight into potential modifications of radical pelvic surgery to improve urinary continence. METHODS: Fresh cadaveric dissections of 12 male hemipelves were undertaken to investigate the neuroanatomy of the urinary rhabdosphincter. RESULTS: Neuroanatomic courses of the nerve supply to the rhabdosphincter revealed that, in the perineum, the perineal nerve (a terminal branch of the pudendal nerve) provided branches directly to the bulbospongiosus muscle and the urinary rhabdosphincter. In the pelvis, the course of the pelvic nerve was as follows: (1) arising from the inferior hypogastric plexus, it had a weblike course beneath the muscle fascia of the levator ani muscle; (2) traveling posterolateral to the rectum, it gave many branches that perforated into the lateral rectum; and (3) at the level of the prostatic apex, still beneath the levator ani muscle fascia (superior fascia), it sent multiple direct branches to the inferolateral aspect of urinary rhabdosphincter. The pudendal nerve traversed the pelvis in the pudendal canal, and, before leaving the pelvis to enter the perineum, it gave an intrapelvic branch that courses with the pelvic nerve to innervate the rhabdosphincter. CONCLUSIONS: Our understanding of the neuroanatomy of what may be the continence nerves has been improved by fresh cadaveric dissection. The rhabdosphincter receives nerve fibers from the pelvic nerve and dual innervation from an intrapelvic branch and a perineal branch of the pudendal nerve. Better understanding of these anatomic findings may have potential surgical significance with respect to improvement in postoperative urinary continence.     

A reporter gene assay for fungal sterol biosynthesis inhibitors

We evaluated the effect of estrogen on nitric oxide (NO)-mediated urethral relaxation in rabbits. Female New Zealand white rabbits, 4-5 weeks old, were treated with 5 mg/kg estradiol dipropionate (estrogen group) or saline (control group) injected intramuscularly weekly for 2 weeks. Electrical field stimulation (supramaximum voltage, 2 ms pulse duration, 0.3-15 Hz and 3 s train) caused frequency-dependent relaxation of urethral strips in both groups, which was inhibited by Nomega-nitro-L-arginine (L.-NNA). This inhibition was overcome by addition of L-arginine. The relaxation induced by nitrergic nerve stimulation was significantly lower in the estrogen group than in the control group. There was no significant difference in sodium nitroprusside-induced urethral relaxation between the two groups. The production of NO in urethral strips during nitrergic nerve stimulation was evaluated by measuring nitrite/nitrate (NO2-/NO3-) levels in both groups, using microdialysis. The NO2-/NO3- production during electrical field stimulation in the estrogen group was significantly less than that in the control group. The NADPH diaphorase-positive reaction in the control group was greater than that in the estrogen group. The results suggest that estrogen treatment may reduce NO synthase activity, and inhibit the relaxation induced by nitrergic nerve stimulation in rabbit urethral smooth muscle.     

Expression of different isoforms of nitric oxide synthase in experimentally denervated and reinnervated skeletal muscle

Denervated muscle fibers express enhanced levels of stress and apoptosis-associated proteins and undergo apoptosis. In experimentally denervated and reinnervated rat facial muscle, we now evaluate changes in the expression patterns of different isoforms of nitric oxide synthase (NOS)-generating nitric oxide (NO), which mediates oxidative stress and apoptosis. Physiological expression of NOS corresponds to a constant sarcolemmal staining pattern for neuronal NOS (nNOS) and a patchy sarcolemmal and weak sarcoplasmic labeling for the endothelial NOS-isoform, with no expression for inducible NOS (iNOS). Denervated muscle displayed distinct downregulation of nNOS with preserved expression of dystrophin. Also, denervated and immediately reinnervated muscle fibers showed decreased expression of nNOS. However, muscle fibers reinnervated for 10 weeks revealed a restored physiological expression of nNOS. There were no changes in the expression of endothelial and inducible NOS. As NO is known to induce growth arrest and collapse of neuronal growth cones, downregulation of NOS may contribute to promotion of axonal regeneration by aiding formation of new endplates. NO is upregulated in reinnervated muscle fibers and thus prevents polyneural hyperinnervation by extrajunctional synapses. Furthermore, downregulation of NOS during denervation is compatible with the finding that low levels of NO contribute to apoptosis instead of necrosis in disease states of oxidative stress.     

Prenatal ultrasonographic findings of intra-abdominal cystic lymphangioma: a case report

The integrity and functional capacity of the urethral sphincter is one of the important prerequisites of urinary incontinence in women. Urodynamic investigations revealed repeatedly that the maximum closure pressure in the median portion of the urethra corresponds to the maximal thickness of the external urethral sphinctor (rhabdosphincter urethrae). This striated muscle is adapted to maintain a relatively steady tonus which assists the closure mechanism of the urethra [4]. In the submitted study the authors focused attention on the ultrasonic visualization of the internal urethral sphincter in order to assess the relationship between the size of this sphincter and the stress type of incontinence (genuine stress incontinence-GSI). The investigation comprised thirty women with confirmed GSI and a control group of thirty asymptomatic volunteers. During perineal ultrasonic examination of women in a supine position by means of an ACUSON 128 XP 10 apparatus using a convex probe with a frequency of 5 MHz the authors recorded statistically significant differences in the areas and maximal thickness of the urethral sphincter in women with stress incontinence and symptom-free women. From the results ensues that the size of this muscle is much smaller in women suffering from GSI.     

Histochemical and immunohistochemical studies on the origin of the blue marlin heater cell phenotype

The superior rectus muscle fibers of marlins, swordfish, sailfish and spearfish are modified for heat production at the expense of contractile ability. Although 'heater cells' are a muscle derivative (Block, 1986, 1991), the myoblast origin and developmental pathway of these thermogenic cells is unknown. To gain insight into heater cell origins, we characterized blue marlin superior rectus muscle and its heater tissue derivative with histochemical and immunological techniques. We specifically employed myosin ATPase and succinate dehydrogenase histochemical assays, and myosin heavy chain immunohistochemistry. Results revealed that marlin superior rectus muscles contain at least six distinct fiber types, and suggested the presence of both twitch and tonic fibers. Immunological results indicate that myosin is present within the thermogenic cells but not in myofibrillar lattices. The antibodies that recognized myosin in heater cells also labeled myosin in the twitch fibers of swimming muscle. In contrast, antibodies that labeled histologically defined tonic fibers did not label heater cells. These results suggest that heater cells and twitch fibers express the same myosin isoform, and establish a phenotypic connection between heater cells and twitch fibers. This conclusion is discussed in the context of the muscle-to-heater trajectory and the muscle fiber-type origin of heater cells.