A pertussis toxin-sensitive G-protein mediates the alpha 2-adrenergic receptor inhibition of melatonin release in photoreceptive chick pineal cell cultures.

The avian pineal gland is a photoreceptive organ that has been shown to contain postjunctional alpha 2-adrenoceptors that inhibit melatonin synthesis and/or release upon receptor activation. Physiological response and [32P]ADP ribosylation experiments were performed to investigate whether pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins) were involved in the transduction of the alpha 2-adrenergic signal. For physiological response studies, the effects of pertussis toxin on melatonin release in dissociated cell cultures exposed to norepinephrine were assessed. Pertussis toxin blocked alpha 2-adrenergic receptor-mediated inhibition in a dose-dependent manner. Pertussis toxin-induced blockade appeared to be noncompetitive. One and 10 ng/ml doses of pertussis toxin partially blocked and a 100 ng/ml dose completely blocked norepinephrine-induced inhibition. Pertussis toxin-catalyzed [32P]ADP ribosylation of G-proteins in chick pineal cell membranes was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Membranes were prepared from cells that had been pretreated with 0, 1, 10, or 100 ng/ml pertussis toxin. In the absence of pertussis toxin pretreatment, two major proteins of 40K and 41K mol wt (Mr) were labeled by [32P]NAD. Pertussis toxin pretreatment of pineal cells abolished [32P] radiolabeling of the 40K Mr G-protein in a dose-dependent manner. The norepinephrine-induced inhibition of both cAMP efflux and melatonin release, as assessed by RIA of medium samples collected before membrane preparation, was also blocked in a dose-dependent manner by pertussis toxin. Collectively, these results suggest that a pertussis toxin-sensitive 40K Mr G-protein labeled by [32P]NAD may be functionally associated with alpha 2-adrenergic signal transduction in chick pineal cells.

Vasoactive intestinal polypeptide and alpha 2-adrenoceptor agonists regulate adenosine 3',5'-monophosphate accumulation and melatonin release in chick pineal cell cultures.

Vasoactive intestinal polypeptide (VIP) has been shown to stimulate melatonin synthesis in mammalian pineal; however, a regulatory role for VIP in the avian pineal has not been explored. Immunocytochemical and physiological response experiments were performed to investigate whether 1) immunoreactive VIP fibers innervated the avian pineal gland; 2) VIP had a specific effect on melatonin release that was mediated by cAMP stimulation; and 3) alpha 2-adrenergic signal transduction was associated with a reduction in cAMP levels. Immunocytochemical experiments demonstrated the presence of both tyrosine hydroxylase- and VIP-immunoreactive fibers in the avian pineal gland. Treatment of dispersed chick pineal cell cultures with VIP stimulated melatonin release (maximum 6-fold increase; EC50 = 1.8 nM) when administered during the 12-h light period of a 12-h light, 12-h dark cycle. Of the other four peptides tested [porcine VIP-(10-28), porcine peptide histidine isoleucine, porcine secretin, and human glucagon), only peptide histidine isoleucine stimulated melatonin release (EC50 = 30 nM). The effect of VIP was mediated by a time- and dose-dependent increase in cAMP accumulation (maximum 4-fold increase). The specific alpha 2-agonist UK-14,304 reduced cAMP accumulation (maximum 43% reduction) and inhibited melatonin release (EC50 = 19 nM) in the presence of 3 X 10(-8) M VIP. Norepinephrine-induced inhibition of nocturnal melatonin release was blocked by the elevation of cAMP achieved through the administration of forskolin (EC50 = 0.2 microM), isobutylmethylxanthine (EC50 = 112 microM), or 8-bromo-cAMP (EC50 = 166 microM). Collectively, these results demonstrate the presence and functional significance of VIP in the avian pineal gland, and the interaction of VIP and norepinephrine at the level of cAMP in the regulation of melatonin biosynthesis.

Pineal melatonin in the Djungarian hamster: photoperiodic regulation of a circadian rhythm.

The pineal melatonin rhythm has been proposed to represent a physiological signal which mediates the effects of photoperiod in a variety of seasonal breeding mammals. Studies in other species have led to the hypothesis that an endogenous circadian mechanism controls pineal melatonin content and can adapt the melatonin rhythm to reflect shifts in the prevailing light-dark schedule. In this report, photoperiodic regulation of the pineal melatonin content was investigated in the male Djungarian hamster, a species that is a long day breeder and photo-periodic from early life. In the first study, a daily pineal melatonin rhythm persisted after 6-8 weeks of constant dark conditions in a specific phase relationship to the onset of wheel running activity. Pineal melatonin levels increased around the time of activity onset, and high values persisted for 14-17 h. In subsequent studies, adult males [previously maintained under long days (16 h of light and 8 h of darkness)] were subjected to one of three conditions: 1) a 3-h advance in the onset of darkness, 2) a delay in the time of light onset, or 3) both alterations in the photoperiod. The acute 3-h advance in onset of the dark period caused an earlier rise in pineal melatonin production by approximately 1.5 h compared to that in controls. In contrast, the falling phase of the pineal melatonin rhythm was not altered by extension of the dark period. Therefore, aspects of the pineal melatonin pattern, i.e. the rising phase and peak duration, reflected changes in the photoperiod, while the falling phase anticipated the onset of the light period predicted by the previous light-dark schedule. These observations support the hypothesis that melatonin production by the pineal is an endogenous circadian rhythm and, as a physiological signal, may convey information about the daylength.

Activity rhythms and photoperiodism of Syrian hamsters in a simulated burrow system.

Male Syrian hamsters were housed in simulated burrows in order to investigate (a) how these nocturnal, fossorial rodents entrain to the prevailing light:dark cycle in this semi-natural habitat and (b) the response of the reproductive system to environmental illumination. The burrow emergence activity of hamsters housed in simulated burrows was compared to the running wheel activity of animals maintained in standard cage conditions. The activity rhythm was similar in both measuring devices. The data suggested that in a natural environment hamsters are only exposed to light for short amounts of time each day. To determine whether brief photoperiodic stimulation could alter the phase angle of entrainment and/or the reproductive condition, burrow housed animals were exposed to a supplemental 30-second light pulse during specified clock hours of the dark period on a daily basis. These light pulses induced a phase shift and maintained a long day reproductive response in what was otherwise a short photoperiod.

Environmental influences upon circadian periodicity of Syrian hamsters.

The free running period (tau) of male Syrian hamsters, when measured as the circadian rhythm of emergence from simulated burrows, was compared to tau when locomotor activity was measured via running wheels. In constant darkness, the inter-individual variability of the tau s was less among burrow housed hamsters than for running wheel housed hamsters. Hence, access to activity wheels eventually led to more disparate activity onset times among the running wheel animals, as compared to the relative population synchrony observed when animals were housed in simulated burrows. When tau s were contrasted between hamsters housed in standard cages with or without running wheels, it appeared that tau values of hamsters housed in simulated burrows were more similar to those of hamsters housed in standard cages without wheels. The data suggests that the tau of the hamster circadian system may be subject to subtle environmental influences.

Role of laparoscopy in the staging of malignant disease.

Although diagnostic laparoscopy has been used by surgeons and gastroenterologists since the early 1900s, today's surgical oncologists have been relatively slow to embrace this technology. Together with the fervor and benefits afforded by laparoscopic therapeutic interventions in the management of patients with benign disease and the diagnostic usefulness in blunt trauma and abdominal pain, awareness has been rekindled regarding the advantages of laparoscopy for the staging of abdominal malignancy. As surgeons begin to realize that extirpative procedures are doomed to failure in curing patients with diffuse abdominal metastases disclosed on laparoscopic assessment, palliative measures, such as stent placement, ablative procedures, balloon dilatation, intraluminal high-dose radiation, and laser techniques will be used commonly by surgical endoscopists and gastroenterologists. Similarly, it is hoped that the use of systemic chemotherapy will achieve better specificity in cell destruction in patients identified laparoscopically to have uncontained disease in the abdominal cavity. The sensitivity of sonography combined with laparoscopy has been shown to approach that of celiotomy in the evaluation of solid organs, thereby avoiding unnecessary laparotomy and its associated morbidities. Using sonography as a complement to laparoscopy will extend the usefulness of both techniques. The application of laparoscopy and the advent of miniaturized laparoscopic instrumentation (Fig. 7), both diagnostic and therapeutic, in the management of patients with abdominal malignancy will be limited only by the creativity and expertise of physicians and instrument makers.

Prospective randomized evaluation of surgical resident proficiency with laparoscopic suturing after course instruction.

BACKGROUND: Laparoscopic suturing is required to develop competency in advanced laparoscopy. METHODS: Manuals detailing laparoscopic suturing were give to 17 Surgery residents. One week later they performed a suture on a training model. Time (s), accuracy (mm), and knot strength (lb) were recorded. The residents were blindly randomized to intervention (n = 9) and control (n = 8) groups. The intervention residents attended a 60-min course with lecture, video, and individual proctoring. Two weeks later they performed a stitch with standard laparoscopic instruments and a stitch with a suturing assist device. Statistical analysis included a Wilcoxon rank-sum test. RESULTS: The intervention residents decreased their suturing time from the first to the second stitich (732.4-257.6s), the control and residents decreased their time from 500.2 s to 421.8 s. The time required to perform the second stitch showed no significant difference between the two groups (p = 0.46), but the difference in reduced time between the first and second stitch was significant (p = 0.001). Using the suturing assist device for the third suture, the intervention and control groups both decreased their times significantly. The control residents performed almost as quickly as the intervention residents with the suturing; device (p = 0.11). Accuracy and knot strength were not different in any test. CONCLUSIONS: Residents can improve suturing skill with a short didactic course and individual proctoring. A suturing assist device decreases time required by inexperienced surgeons to device perform an intracorporeal tie.

Laparoscopic vs open resection of gastric stromal tumors.

BACKGROUND: Gastric stromal tumors are rare neoplasms that may be benign or malignant. Given that malignant gastric stromal tumors rarely involve lymph nodes and require excision with negative margins, they appear amendable to laparoscopic excision. There are few reports of laparoscopic resection, and no comparisons have been done between laparoscopic and open surgery. This study compares the relative efficacy of the two approaches. METHODS: Between May 1994 and December 2000, 33 patients underwent 35 operations for gastric stromal tumors. Laparoscopic resections were performed in 21 patients; open resections were done in 12 patients. The medical records of the patients were reviewed retrospectively with regard to operating time, blood loss, length of stay, and clinical course. RESULTS: Patient demographics, tumor characteristics (mean tumor size, benign vs malignant), and presenting symptoms were similar for both groups. In the laparoscopic group, 15 wedge resections; three partial gastrectomies, and three transgastric needlescopic enucleations were performed. In the open group, six wedge resections, four antrectomies, and two partial proximal gastrectomies were performed. There were no significant differences in mean operative time (169 vs 160 min), mean estimated blood loss (106 vs 129 cc), or perioperative complication rate (9.5% vs 8.3%) between the laparoscopic and open groups, respectively. The mean length of stay was significantly less (p<0.05) in the laparoscopic group (3.8 vs 6.2 days). Average follow-up was 1.5 years. One patient in each group has died due to metastatic disease. There have been no trocar site recurrences. CONCLUSIONS: Laparoscopic resection of gastric stromal tumors is safe and appropriate. Tumor size, operating time, and estimated blood loss were equivalent to the open approach, and there was a statistically shorter hospital stay in the laparoscopic group.

Effectiveness of the ultrasonic coagulating shears, LigaSure vessel sealer, and surgical clip application in biliary surgery: a comparative analysis.

Advancements in laparoscopic surgery are often dictated by the limitations of technical instrumentation. Energy sources other than electrosurgery have become popular with the promise of quick and effective vascular control. With their success surgeons have begun using these on structures other than blood vessels with little or no data establishing their efficacy or safety. This study evaluates alternative energy sources in sealing ductal structures for possible use in liver or gallbladder surgery. After elective cholecystectomy cystic ducts (n = 45) were resealed ex vivo with surgical clips (n = 14), ultrasonic coagulating shears (n = 16), or electrothermal bipolar vessel sealer (n = 15), and bursting pressures were measured. Nineteen additional human cystic ducts were randomized to seal by ultrasonic coagulating shears (n = 9) or electrothermal bipolar vessel sealer (n = 10) and fixed in 10 per cent buffered formalin for histologic evaluation of thermal spread (mm). After this nine adult pigs were randomized to laparoscopic ligation and transection of the common bile duct using surgical clips (n = 3), ultrasonic coagulating shears (n = 3), or electrothermal bipolar vessel sealer (n = 3). The animals underwent necropsy for assessment of seal integrity on the sixth postoperative day. In the ex vivo study the mean cystic duct bursting pressure was 621 mm Hg with surgical clips and 482 mm Hg with the electrothermal bipolar vessel sealer (P = 0.39). The mean cystic duct bursting pressure after ultrasonic coagulating shears was 278 mm Hg, which was statistically less than surgical clips (P = 0.007) and electrothermal bipolar vessel sealer (P = 0.02). The mean thermal spread was 3.5 mm for ultrasonic coagulating shears and 13.4 mm for electrothermal bipolar vessel sealer (P = 0.0002). All animals undergoing ligation and transection of the common bile duct with ultrasonic coagulating shears and electrothermal bipolar vessel sealer developed bile peritonitis by postoperative day 6 as a result of seal leak. All animals undergoing surgical clip ligation and transection of the common bile duct maintained seal integrity. The mean common bile duct pressure above the surgical clip was 12 mm Hg (range 10-14). In conclusion the acute ex vivo study demonstrated a significant difference in the cystic duct bursting pressure between surgical clips and ultrasonic coagulating shears and between electrothermal bipolar vessel sealer and ultrasonic coagulating shears. The ultrasonic coagulating shears and electrothermal bipolar vessel sealer failed to maintain seal integrity in the in vivo animal study. Given the failure of the ultrasonic coagulating shears and electrothermal bipolar vessel sealer in the animal model these energy sources should not be used for transection of the cystic duct or major hepatic ducts during hepatobiliary surgery.

Maternal influence on activity rhythms and reproductive development in Djungarian hamster pups.

Photoperiodism and entrainment of the circadian rhythm of locomotor activity were investigated in juvenile Djungarian hamsters. Animals were housed in simulated burrows. Activity was measured as the animal's emergence from a dark nest chamber into an outer box exposed to the room illumination. This burrow emergence activity exhibited marked circadian rhythmicity. Interactions between mother hamsters and their offspring were examined in the simulated burrow system. Male reproductive responses were determined by measuring testicular weights at the time of weaning. It was shown that photoperiodic information received between Days 1 and 15 of life failed to alter the rate of testicular development, but that after Day 15 testicular growth was photoperiod-dependent. The mother, when entrained to a long photoperiod, did not influence the photoperiodic responses of her pups when they were confined to a dark nest box. In contrast, the mother did influence the circadian entrainment patterns of her pups. Pups exhibited a well-developed circadian activity rhythm at weaning with a phase angle roughly similar to that of the mother's activity rhythm. When the maternal rhythms were discrepant with photoperiod information received by the pups directly from the environment, the pups' activity rhythms were synchronized with the light/dark cycle rather than with the rhythm of their mother. Thus, it appears that although pups may first become entrained by maternal cues, they rapidly adjust to the environmental light cycle after leaving the nest.

Alpha-2 adrenergic regulation of melatonin release in chick pineal cell cultures.

The chick pineal gland expresses a circadian rhythm of melatonin biosynthesis, with elevated levels at night and low levels during the day. The rhythm of melatonin is regulated both by circadian oscillators located within the gland itself and by adrenergic input from the sympathetic nervous system. Previous work has shown that norepinephrine administration inhibits melatonin biosynthesis, as measured by the activity of the enzyme serotonin N-acetyltransferase. As a first step toward understanding the mechanisms by which norepinephrine regulates melatonin production in the chick pineal, we have identified the adrenergic receptor involved. Dissociated chick pineal cell cultures were prepared and melatonin release was measured on days 5 and 6 of culture using radioimmunoassay. The effects of adrenergic agonists and antagonists on the nocturnal increase of melatonin release during the 12 hr dark portion of a LD12:12 light cycle were determined. Norepinephrine inhibited melatonin release in a dose-dependent manner, with an average EC50 of 19.7 nM +/- 2.23 (SEM). Melatonin release values ranged from 100 to 4% of the level seen in control cultures, depending on the dose of norepinephrine. The physiological response to epinephrine, norepinephrine, and isoproterenol was stereospecific. The (-) stereoisomer was 6, 8, and 37 times more potent than the (+) stereoisomer, respectively. EC50 values (in nM) for adrenergic agonists were as follows: alpha-methyl-(-)-norepinephrine, 2.46; tramazoline, 3.06; guanabenz, 3.31; clonidine, 3.70; oxymetazoline, 4.29; (-)-epinephrine, 7.44; (-)-norepinephrine, 19.7; (-)-isoproterenol, 463; and (-)-phenylephrine, 659. Schild analysis was used to determine the relative potency of adrenergic antagonists. pA2 values for adrenergic antagonists were as follows: rauwolscine, 9.55; RX78 1094, 8.32; yohimbine, 8.14; phentolamine, 7.11; prazosin, 5.93; and (-)-propranolol, less than 6. The relative potencies of both adrenergic agonists and antagonists demonstrate that alpha-2 receptors mediate norepinephrine-induced inhibition of melatonin release in chick pineal cell cultures. The identification of alpha-2 receptors in chick pineal cells should aid in our understanding of the biochemical events initiated by receptor activation that regulate melatonin synthesis.

Strategies for laparoscopic diagnosis of malignancy.

Accurate cancer diagnosis and staging are crucial to the determination of an efficacious treatment plan for localized and advanced malignancy. The physician must differentiate patients with potentially resectable, localized disease from those with advanced and/or distant disease. The diagnostic and staging modalities currently available are expensive and often inaccurate. This can result in the nonoperative management of potentially resectable malignancies or, more commonly, in an underestimation of the preoperative cancer stage with intraoperative evidence of advanced/metastatic disease. The combination of laparoscopy and laparoscopic ultrasonography can be used to help diagnose and stage malignancies and select patients for either curative or palliative procedures.