RESUMEN
Memory consolidation is associated with sleep physiology but the contribution of specific sleep stages remains controversial. To clarify the contribution of REM sleep, participants were administered two REM sleep-sensitive tasks to determine if associated changes occurred only in REM sleep. Twenty-two participants (7 men) were administered the Corsi Block Tapping and Tower of Hanoi tasks prior to and again after a night of sleep. Task improvers and non-improvers were compared for sleep structure, sleep spindles, and dream recall. Control participants (N = 15) completed the tasks twice during the day without intervening sleep. Overnight Corsi Block improvement was associated with more REM sleep whereas Tower of Hanoi improvement was associated with more N2 sleep. Corsi Block improvement correlated positively with %REM sleep and Tower of Hanoi improvement with %N2 sleep. Post-hoc analyses suggest Tower of Hanoi effects-but not Corsi Block effects-are due to trait differences. Sleep spindle density was associated with Tower of Hanoi improvement whereas spindle amplitude correlated with Corsi Block improvement. Number of REM awakenings for dream reporting (but not dream recall per se) was associated with Corsi Block, but not Tower of Hanoi, improvement but was confounded with REM sleep time. This non-replication of one of 2 REM-sensitive task effects challenges both 'dual-process' and 'sequential' or 'sleep organization' models of sleep-dependent learning and points rather to capacity limitations on REM sleep. Experimental awakenings for sampling dream mentation may not perturb sleep-dependent learning effects; they may even enhance them.
Asunto(s)
Consolidación de la Memoria/fisiología , Recuerdo Mental/fisiología , Sueño REM/fisiología , Adulto , Encéfalo/fisiología , Sueños/fisiología , Electroencefalografía , Femenino , Humanos , Masculino , Pruebas Neuropsicológicas , Fases del Sueño , Adulto JovenRESUMEN
The effects of divalent cations on the E-4031-sensitive repolarization current (I(Kr)) were studied in single ventricular myocytes isolated from rabbit hearts. One group of divalent cations (Cd2+, Ni2+, Co2+, and Mn2+) produced a rightward shift of the I(Kr) activation curve along the voltage axis, increased the maximum I(Kr) amplitude (i.e., relieved the apparent inward rectification of the channel), and accelerated I(Kr) tail current kinetics. Another group (Ca2+, Mg2+ and Sr2+) had relatively little effect on I(Kr). The only divalent cation that blocked I(Kr) was Zn2+ (0.1-1 mM). Under steady-state conditions, Ba2+ caused a substantial block of I(K1) as previously reported. However, block by Ba2+ was time dependent, which precluded a study of Ba2+ effects on I(Kr). We conclude that the various effects of the divalent cations can be attributed to interactions with distinct sites associated with the rectification and/or inactivation mechanism of the channel.
Asunto(s)
Antiarrítmicos/farmacología , Proteínas de Transporte de Catión , Cationes Bivalentes/farmacología , Potenciales de la Membrana/efectos de los fármacos , Piperidinas/farmacología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/fisiología , Piridinas/farmacología , Animales , Bario/farmacología , Cadmio/farmacología , Calcio/farmacología , Células Cultivadas , Cobre/farmacología , Canales de Potasio Éter-A-Go-Go , Ventrículos Cardíacos , Cinética , Magnesio/farmacología , Manganeso/farmacología , Potenciales de la Membrana/fisiología , Níquel/farmacología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Conejos , Estroncio/farmacologíaRESUMEN
We have measured the E-4031-sensitive repolarization current (IKr) in single ventricular myocytes isolated from rabbit hearts. The primary goal of this analysis was a description of the IKr kinetic and ion transfer properties. Surprisingly, the maximum time constant of this component was 0.8 s at 33-34 degrees C, which is significantly greater than the value of 0.18 s previously reported under similar conditions in the original measurements of IKr from guinea pig ventricular myocytes. The primary, novel feature of our analysis concerns the relationship of the bell-shaped curve that describes the voltage dependence of the kinetics and the sigmoidal curve that describes the activation of IKr. The midpoint of the latter occurred at approximately +10 mV on the voltage axis, as compared to -30 mV for the point on the voltage axis at which the maximum time constant occurred. Moreover, the voltage dependence of the kinetics was much broader than the steepness of the activation curve would predict. Taken together, these results comprise a gating current paradox that is not resolved by the incorporation of a fast inactivated state in the analysis. The fully activated current-voltage relation for IKr exhibited strong inward-going rectification, so much so that the current was essentially nil at +30 mV, even though the channel opens rapidly in this voltage range. This result is consistent with the lack of effect of E-4031 on the early part of the plateau phase of the action potential. Surprisingly, the reversal potential Of /Kr was ~15 mV positive to the potassium ion equilibrium potential,which indicates that this channel carries inward current during the latter part of the repolarization phase of the action potential.
Asunto(s)
Miocardio/metabolismo , Potasio/metabolismo , Animales , Antiarrítmicos/farmacología , Fenómenos Biofísicos , Biofisica , Resistencia a Medicamentos , Cobayas , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Técnicas In Vitro , Activación del Canal Iónico , Transporte Iónico/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Modelos Biológicos , Piperidinas/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Piridinas/farmacología , ConejosRESUMEN
The primary structure of bovine cathepsin S was determined by combining results of protein and peptide sequencing with the sequence deduced from nucleic acid sequencing. Using polymerase chain reaction (PCR) technology, cDNA clones commencing at amino acid 22 of the mature enzyme and continuing through the 3' untranslated region of bovine cathepsin S mRNA were isolated and sequenced. The open reading frame in these overlapping clones correctly predicts the determined amino acid sequence of 13 tryptic peptides derived from purified bovine spleen cathepsin S. The deduced amino acid sequence shows that mature bovine cathepsin S consists of 217 amino acids corresponding to a molecular weight of 23.7 kDa. Cathepsin S belongs to the papain superfamily of lysosomal cysteine proteinases and shares 41% identity with papain. Amino acid sequence identities of bovine cathepsin S to human cathepsins L, H, and B are 56%, 47% and 31% respectively.
Asunto(s)
Catepsinas/genética , Cisteína Endopeptidasas , Endopeptidasas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Catepsina B/química , Catepsina H , Catepsina L , Catepsinas/química , Bovinos , Humanos , Datos de Secuencia Molecular , Papaína/química , Conformación Proteica , Alineación de SecuenciaRESUMEN
Using cultured islet cells from both splenic lobe (SL) and uncinate process (UP) of the dog pancreas, we have measured responses of insulin, glucagon, and pancreatic polypeptide (PP) to glucose, carbamylcholine (carbachol), and exogenous PP. The results show that both insulin and PP cells of the two developmentally distinct lobes of the pancreas respond differentially to secretagogues. The results suggest that PP may play a role in regulation of islet cell secretion. Secretion of insulin by SL and UP cultures in response to 28 mM glucose in a 2h incubation was significantly greater, 2.9- (p less than 0.01) and 1.5-fold (p less than 0.01), respectively, than secretion by respective control cultures at 2.8 mM glucose. The difference in degree of stimulation between SL and UP was also significant (p less than 0.02). At 2.8 mM glucose, SL and UP cultures secreted 10% and 8.8%, respectively, of immunoreactive insulin (IRI) contents of the cultured cells (NS). Dose-response curves showed that for up to 8.5 mM glucose the degree of stimulation of SL was no greater than UP, but the UP response had nearly plateaued; whereas, the SL response continued to increase, such that SL was greater than UP at 16.7 mM glucose (p less than 0.01). Secretion of PP by SL and UP cultures in response to 5 microM carbachol and 2.8 mM glucose in a 2-h incubation was significantly greater, 2.2- (p less than 0.002) and 3.9-fold (p less than 0.002), respectively, than secretion by respective control cultures without carbachol. The difference in degree of stimulation between SL and UP was also significant (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Carbacol/farmacología , Glucosa/farmacología , Islotes Pancreáticos/efectos de los fármacos , Polipéptido Pancreático/farmacología , Animales , Células Cultivadas , Perros , Relación Dosis-Respuesta a Droga , Glucagón/farmacología , Insulina/metabolismo , Insulina/farmacología , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Polipéptido Pancreático/metabolismo , RadioinmunoensayoRESUMEN
In this study, we found that the ratio of proinsulin to total immunoreactive insulin was much higher in 22 patients with Type 2 (non-insulin-dependent) diabetes mellitus than in 28 non-diabetic control subjects of similar age and adiposity (32 +/- 3 vs 15 +/- 1%, p less than 0.001). In addition, the arginine-induced acute proinsulin response to total immunoreactive insulin response ratio was greater in diabetic patients (n = 10) than in control subjects (n = 9) (8 +/- 2 vs 2 +/- 0.5%, p = 0.009), suggesting that increased islet secretion per se accounted for the increased ratio of proinsulin to immunoreactive insulin. One explanation for these findings is that increased demand for insulin in the presence of islet dysfunction leads to a greater proportion of proinsulin secreted from the B cell. We tested this hypothesis by comparing proinsulin secretion before and during dexamethasone-induced insulin resistance in diabetic patients and control subjects. Dexamethasone treatment (6 mg/day for 3 days) raised the proinsulin to immunoreactive insulin ratio in control subjects from 13 +/- 2 to 21 +/- 2% (p less than 0.0001) and in diabetic patients from 29 +/- 5 to 52 +/- 7% (p less than 0.001). Dexamethasone also raised the ratio of the acute proinsulin response to the acute immunoreactive insulin response in control subjects from 2 +/- 0.5 to 5 +/- 2% (p = 0.01) and in diabetic patients from 8 +/- 2 to 14 +/- 4% (p = NS), suggesting that the dexamethasone-induced increment in the basal ratio of proinsulin to immunoreactive insulin was also due to increased secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Resistencia a la Insulina , Insulina/sangre , Proinsulina/sangre , Arginina , Glucemia/metabolismo , Dexametasona , Diabetes Mellitus/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , ObesidadRESUMEN
We compared the effects of sympathetic nerve stimulation to that of pancreatic norepinephrine (NE) infusion on pancreatic somatostatin-like immunoreactivity (SLI) and pancreatic polypeptide (PP) secretion in the halothane-anesthetized dog. During electrical stimulation (8 Hz, 1 msc, 10 mA, n = 6) of the sympathetic nerves surrounding the pancreatic artery, the pancreatic SLI output decreased from 827 +/- 340 fmol/min to 231 +/- 47 fmol/min (delta = -596 +/- 217 fmol/min, P less than 0.05) after 5 min, and PP output decreased from 11,972 +/- 374 pg/min to 5,518 +/- 774 pg/min (delta = -6,454 +/- 1,932 pg/min, P less than 0.02) after 3 min of stimulation. Arterial SLI or PP levels did not change. Sympathetic nerve stimulation also decreased pancreatic blood flow and increased pancreatic venous NE levels. To determine whether the NE, released locally during sympathetic nerve stimulation, is responsible for this inhibition of pancreatic SLI and PP outputs, exogenous NE was infused into the pancreatic artery at three different dose levels. The low dose of 12 ng/min (n = 6) increased pancreatic venous NE levels like sympathetic nerve stimulation. The medium dose of 120 ng/min (n = 6) reproduced the nerve stimulation-induced decrease of pancreatic blood flow. The high dose of 1,200 ng/min (n = 6) markedly exceeded both. It was found that neither the low nor the medium infusion rates of NE significantly affected pancreatic SLI or PP output. In contrast, infusion of NE at the very high dose level of 1,200 ng/min decreased pancreatic SLI output from 850 +/- 165 fmol/min to 318 +/- 59 fmol/min after 5 min of infusion (delta = -532 +/- 143 fmol/min, P less than 0.01) and decreased PP secretion from 22,777 +/- 7,082 pg/min to 12,764 +/- 6,100 pg/min after 3 min of infusion (delta = -10,013 +/- 2,399 pg/min, P less than 0.01). During this high dose rate NE infusion, both the increment of pancreatic venous SPV levels of NE and the decrement of pancreatic blood flow markedly exceeded the effects produced by sympathetic nerve stimulation. We conclude from this study in dogs: that selective electrical stimulation of the sympathetic nerves entering the pancreas decreases blood flow and inhibits pancreatic SLI and PP secretion, that NE infused into the pancreatic artery at moderate rates reproduces the decrease in blood flow yet does not reproduce the inhibition of pancreatic SLI and PP secretion, and that extremely high concentrations of NE, which markedly restrict pancreatic blood flow, decrease both pancreatic SLI and PP outputs.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Norepinefrina/farmacología , Páncreas/metabolismo , Polipéptido Pancreático/metabolismo , Somatostatina/metabolismo , Sistema Nervioso Simpático/fisiología , Animales , Arterias , Perros , Estimulación Eléctrica , Cinética , Norepinefrina/sangre , Páncreas/irrigación sanguínea , Páncreas/inervación , Flujo Sanguíneo Regional/efectos de los fármacos , VenasRESUMEN
The structure of the canine prepropancreatic polypeptide (preproPP) cDNA was determined. The nucleotide sequence conservation between human and canine preproPP is very high for the signal peptide (82%) and the region coding for the 36 amino acid pancreatic polypeptide (PP) (92%). The overall sequence homology for the C-terminal portion of proPP containing the icosapeptide and a C-terminal extension peptide is only 63% whereas the 3'-untranslated regions of human and canine PP mRNA share 73% homology after alignment for maximal homology. The only sequence conservation in icosapeptide is the region coding for the last 10 amino acids of the icosapeptide. Comparison of PP immunoactivity and PP mRNA concentrations in extracts of the developmentally distinct uncinate process and splenic lobes of the canine pancreas revealed the same ratio of mRNA concentrations (16 +/- 6.5) and PP peptide concentrations (18 +/- 7.0) in the uncinate process compared to the splenic lobe (n = 6). However, a similar comparison of insulin C-peptide (CP) immunoactivity and insulin mRNA concentration revealed a smaller ratio of CP immunoactivity (0.37 +/- 0.05) than insulin mRNA (0.58 +/- 0.10) between the same lobes (P less than 0.0074, n = 6). This increased steady state CP concentration relative to insulin mRNA in splenic lobe compared to the uncinate process was not observed for PP peptide and mRNA.
Asunto(s)
ADN/genética , Insulina/genética , Páncreas/metabolismo , Polipéptido Pancreático/genética , ARN Mensajero/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Perros , Biblioteca de Genes , Humanos , Islotes Pancreáticos/metabolismo , Datos de Secuencia Molecular , Precursores de Proteínas/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
To determine the suitability of halothane anesthesia for studies of parasympathetic control of the endocrine pancreas in dogs, we assessed the effect of halothane on reflex, direct neural, and direct chemical activation of the parasympathetic input to the islet. Levels or output of pancreatic polypeptide (PP), an islet hormone under predominant cholinergic influence, were used as an indicator of the degree of parasympathetic activation and its potential suppression by anesthesia. Reflex stimulation of the parasympathetic nervous system by 2-deoxy-D-glucose (2-DG) in dogs anesthetized with halothane (0.8%) caused a fourfold increase in plasma PP levels, equivalent to the response in conscious dogs. In contrast, 2-DG at this dose and at a threefold higher dose did not alter PP levels in dogs anesthetized with pentobarbital (30 mg/kg iv), suggesting that halothane at this dose is not suppressive and that pentobarbital is very suppressive on reflex activation of the parasympathetic nerves to the pancreas. Bilateral electrical stimulation of the cervical vagi in halothane-anesthetized dogs elicited a sixfold increase in the pancreatic output of PP. The same stimulation caused only a twofold increase of PP output in pentobarbital-anesthetized dogs. These data suggests that halothane is also less inhibitory than pentobarbital on either peripheral neurotransmission or pancreatic F-cell responsiveness. The effect of direct activation of the F-cell by bethanechol did not differ between the two anesthesias. Therefore, the attenuated PP response to vagal stimulation in pentobarbital-anesthetized dogs is probably due to an action of pentobarbital on peripheral neurotransmission, perhaps at the intrapancreatic parasympathetic ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Halotano/farmacología , Islotes Pancreáticos/efectos de los fármacos , Sistema Nervioso Parasimpático/efectos de los fármacos , Pentobarbital/farmacología , Reflejo/efectos de los fármacos , Anestesia General , Animales , Desoxiglucosa/farmacología , Depresión Química , Perros , Estimulación Eléctrica , Islotes Pancreáticos/inervación , Polipéptido Pancreático/metabolismo , Sistema Nervioso Parasimpático/fisiología , Transmisión Sináptica/efectos de los fármacos , Nervio Vago/fisiologíaRESUMEN
In this assay for immunoreactive human proinsulin (IRPI), it first is separated from plasma by use of an antiserum to human C-peptide. An immunoprecipitate is then formed by using a precipitating antiserum and polyethylene glycol, after which IRPI is dissociated from the antiserum by incubation in warm HCl, pH 2.0. The resulting mixture is assayed for insulin immunoreactivity by a double-antibody tracer-competition method involving incubation for four days with a high-affinity anti-insulin antiserum. Human proinsulin of recombinant-DNA origin is used as the standard. Added C-peptide at supraphysiological concentrations did not interfere with or react in the assay. Human insulin cross reacted by 1.5%. The detection limit for IRPI (2 SD from zero-dose binding) is 3 pmol/L. Proinsulin conversion intermediates are measured nearly as well as intact proinsulin. IRPI concentrations in 10 nondiabetic human subjects averaged 12.0 (SEM 1.6) pmol/L. The ratio of proinsulin to immunoreactive insulin averaged 14.3 (SEM 2.2)%. After intravenous arginine, the increase in proinsulin was less than that of insulin, and it declined more slowly.
Asunto(s)
Péptido C/inmunología , Insulina/inmunología , Proinsulina/análisis , Arginina , Reacciones Cruzadas , Diabetes Mellitus/sangre , Humanos , Sueros Inmunes , Matemática , RadioinmunoensayoRESUMEN
Techniques of in vitro receptor autoradiography were used to visualize binding of 125I-insulin on slices of frozen rat brain. Slide-mounted sections of frozen rat brain were incubated in 0.05 nM porcine 125I-monoiodoinsulin, alone or mixed with 1 microM unlabeled porcine insulin, ribonuclease, or glucagon, for 2 h at 22 degrees C. The labeled brain slices were apposed to LKB Ultrofilm to generate autoradiograms. The method permitted equal access of labeled insulin to both sides of the blood-brain barrier and localization of insulin binding sites in small anatomic regions. Quantitative estimates of specific iodoinsulin binding were made by computer digital image densitometry of the autoradiographic film images. High concentrations of specific binding sites for iodoinsulin were present in the choroid plexus of the lateral (26.9 +/- 2.0 X 10(-3) fmol/mm2), fourth (18.3 +/- 3.0 X 10(-3) fmol/mm2), and third (13.2 +/- 1.5 X 10(-3) fmol/mm2) ventricles (insulin binding is expressed per unit area of autoradiographic image). Binding to the third ventricular choroid plexus was similar to the concentrations observed for liver slices and the external plexiform layer of the olfactory bulb. Specific binding of iodoinsulin in the cingulate cortex and other surrounding regions was less than in choroid plexus. Ribonuclease or glucagon had no measurable effect on binding when mixed with labeled insulin. The results support the hypothesis that the choroid plexus has a high density of receptors for insulin, and suggests that the choroid plexus may be a target of CSF insulin action and/or a site of insulin transport into the CSF.
Asunto(s)
Plexo Coroideo/metabolismo , Receptor de Insulina/metabolismo , Animales , Autorradiografía , Barrera Hematoencefálica , Glucagón/metabolismo , Técnicas In Vitro , Insulina/metabolismo , Masculino , Ratas , Ribonucleasas/metabolismoRESUMEN
Both a high physiological concentration (13.1 nM) of epinephrine (E) and acute exercise (AEx) have previously been shown to increase 125I-insulin binding in skeletal muscle. To investigate the site and mechanism of the effect of epinephrine on binding and the possible link between epinephrine- and AEx-enhanced insulin binding, we measured insulin binding in three different preparations: 1) crude membranes derived from whole soleus muscle incubated in vitro with 13.1 nM E, 2) crude membranes with E present in the binding assay, and 3) purified plasma membranes with E present. Epinephrine enhanced binding in all three preparations by 169, 144, and 164%, respectively, at low concentrations of insulin but had little effect at high concentrations. Epinephrine, therefore appears to have its effect at the plasma membrane. Propranolol (10 microM), a beta-adrenergic antagonist, blocked E-enhanced insulin binding and when added to crude membranes made from soleus and extensor digitorum longus muscle of AEx rats reversed the increase in binding seen with exercise. This indicates that E-enhanced insulin binding is mediated by beta-adrenergic receptors and that AEx enhances insulin binding via beta-adrenergic receptors. Sodium orthovanadate (3 mM), a phosphotyrosyl-protein phosphatase inhibitor, also inhibited the increase in insulin binding due to E, implying that E may increase insulin binding by activating a phosphotyrosyl-protein phosphatase which decreases the phosphorylation of a plasma membrane protein, presumably the insulin receptor.
Asunto(s)
Epinefrina/farmacología , Insulina/metabolismo , Músculos/metabolismo , Simpatomiméticos/farmacología , Animales , Membrana Celular/metabolismo , Epinefrina/metabolismo , Masculino , Membranas/metabolismo , Esfuerzo Físico , Propranolol/farmacología , Ratas , Ratas Endogámicas , Factores de Tiempo , Tritio , Vanadatos , Vanadio/farmacologíaRESUMEN
We measured insulin binding to crude membranes from rat skeletal muscle, with binding expressed relative to the sarcolemmal marker, cholesterol ester (CE). The amount of CE in the sarcolemma remained constant after streptozotocin-induced diabetes and acute exercise (swam for 90 min). Soleus (predominantly slow-twitch fibers) had higher insulin binding capacity than extensor digitorum longus (predominantly fast twitch). Both diabetes and acute exercise enhanced insulin binding. The shape of the enhanced insulin binding curve differed, however, between diabetes and acute exercise. Diabetes elicited a uniform increase in binding across the insulin concentrations measured (0.04-166 nM); acute exercise elicited the largest increase at the lower concentrations, suggesting different mechanisms cause the enhanced binding. Addition of 13.1 nM epinephrine to the perfusate in a rat hindlimb preparation increased insulin binding in a pattern similar to acute exercise. In contrast, muscular contraction stimulated by the sciatic nerve (1 Hz) or reduction of perfusate insulin from 100 to 40 pM, two additional correlates of acute exercise, had no effect. The increased insulin binding after acute exercise, therefore, appears to be mediated through elevated levels of catecholamines and not upregulation of the insulin receptor.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Epinefrina/farmacología , Insulina/metabolismo , Músculos/metabolismo , Esfuerzo Físico , Animales , Ésteres del Colesterol/metabolismo , Masculino , Contracción Muscular , Ratas , Ratas Endogámicas , Sarcolema/metabolismo , Estreptozocina , Fracciones Subcelulares/metabolismo , Factores de TiempoRESUMEN
To investigate the effect of vagal nerve stimulation on the release of pancreatic somatostatin, we electrically stimulated (10 Hz, 5 ms, 13.5 mA, and 10 min) the thoracic vagi just below the heart in halothane anesthetized dogs (n = 15). The stimulation increased the pancreatic output of somatostatinlike immunoreactivity (SLI) (delta = +248 +/- 81 fmol/min, P less than 0.005; base-line levels = 455 +/- 150 fmol/min). min). Arterial plasma SLI levels increased as well (delta = +16 +/- 3 fmol/ml, P less than 0.001; base-line levels = 65 +/- 3 fmol/ml), reflecting stimulation of extrapancreatic SLI secretion. Significant vagal activation was verified by a fivefold increase of pancreatic output of pancreatic polypeptide (PP) (delta = +31.4 +/- 5.9 ng/min, P less than 0.001; base-line levels = 7.8 +/- 0.9 ng/min). Atropine pretreatment (n = 6) inhibited partially both the PP response (delta = +7.9 +/- 3.8 ng/min after atropine) and the pancreatic SLI response (delta = +92 +/- 29 fmol/min) to vagal nerve stimulation. However, atropine pretreatment did not modify the arterial SLI response (delta = +20 +/- 7 fmol/ml). Hexamethonium pretreatment (n = 9) completely abolished all three responses. We conclude that 1) electrical stimulation of the vagus stimulates pancreatic SLI, extrapancreatic SLI, and PP release in vivo in the dog; 2) both muscarinic and nonmuscarinic mechanisms mediate the PP and pancreatic SLI responses; 3) a nonmuscarinic mechanism mediates the extrapancreatic SLI response; and 4) all three responses are mediated via ganglionic nicotinic receptors.
Asunto(s)
Somatostatina/metabolismo , Nervio Vago/fisiología , Animales , Atropina/farmacología , Fenómenos Biomecánicos , Perros , Estimulación Eléctrica , Sistema de Conducción Cardíaco , Hexametonio , Compuestos de Hexametonio/farmacología , Cuello/inervación , Páncreas/metabolismo , Polipéptido Pancreático/metabolismo , Tórax/inervaciónRESUMEN
We sought to determine in overnight-fasted dogs whether basal or 2-deoxy-D-glucose (2-DG)-stimulated levels of pancreatic polypeptide (PP) could be reliably used as an index of cholinergic activity at the pancreas and thereby determine the effect of pentobarbital anesthesia on this cholinergic outflow. At low basal PP levels, either atropine or pentobarbital had a small effect on PP levels; at higher basal levels, both atropine and pentobarbital had a larger effect. Thus both drugs decreased PP in proportion to its initial basal level, suggesting that basal PP levels have a variable cholinergic component. Atropine abolished the PP response to intravenous 2-DG, confirming in our animal model that the PP response to neuroglucopenia is entirely cholinergically mediated. Pentobarbital also abolished the PP response to 2-DG, suggesting that anesthesia either suppresses cholinergic outflow to the pancreas or the response of the pancreatic F-cell to it. To test the latter hypothesis, the acetylcholine analogue bethanechol was administered before and during pentobarbital anesthesia. The PP response to direct cholinergic stimulation was not abolished by pentobarbital, although it was reduced modestly. We conclude that only part of the basal level of PP is under cholinergic control; all of the PP response to 2-DG is cholinergically mediated; pentobarbital anesthesia abolishes the cholinergic input to the pancreas; and if the endogenous cholinergic input influences certain pancreatic endocrine and exocrine responses, then its contribution would be seriously underestimated when studied in pentobarbital-anesthetized animals.
Asunto(s)
Polipéptido Pancreático/metabolismo , Pentobarbital/farmacología , Animales , Atropina/farmacología , Betanecol , Compuestos de Betanecol/farmacología , Desoxiglucosa/farmacología , Perros , Relación Dosis-Respuesta a Droga , Páncreas/efectos de los fármacos , Páncreas/fisiología , Polipéptido Pancreático/sangre , Sistema Nervioso Parasimpático/efectos de los fármacos , Receptores Colinérgicos/fisiología , Receptores Muscarínicos/fisiología , Reflejo/efectos de los fármacos , VagotomíaRESUMEN
Insulin binding was measured in membrane particles prepared from the liver and several brain regions of 4-month-old female Zucker fa/fa (obese), Fa/fa (heterozygous), and Fa/Fa (lean) rats. High affinity insulin binding was decreased in the olfactory bulb of fatty (0.23 pmol bound/mg protein) and heterozygous (0.16 pmol/mg) rats compared with that in the lean controls (0.64 pmol/mg). Total binding was not changed in the cerebral cortex or hypothalamus. High affinity insulin binding was also decreased in the liver of both fatty (0.44 +/- 0.22 pmol/mg; P less than 0.01) and heterozygous (0.75 +/- 0.35 pmol/mg) animals compared with that in the lean rats (2.10 +/- 1.55 pmol/mg). This decreased binding is probably not due to down-regulation of receptors in the heterozygous rats, as they do not exhibit the hyperinsulinemia observed in the fatty rats. Rather, our findings suggest that there is a gene-related alteration in insulin binding in the Zucker rat, as low binding was observed in rats carrying either one (Fa/fa) or two (fa/fa) doses of the gene. We postulate that this central defect in insulin binding may contribute to inadequate perception of a central insulin feedback signal and to the hyperphagia observed in the obese rats.
Asunto(s)
Encéfalo/metabolismo , Genes , Insulina/metabolismo , Hígado/metabolismo , Ratas Mutantes/genética , Ratas Zucker/genética , Animales , Sitios de Unión , Femenino , Heterocigoto , Obesidad/metabolismo , Bulbo Olfatorio/metabolismo , Ratas , Ratas Zucker/metabolismo , Distribución TisularRESUMEN
Insulin levels increase after 2-deoxyglucose (2DG) administration in dogs. This observation is in contrast to the decrease in insulin level post-2DG in baboons and rabbits. To evaluate a possible neural mechanism mediating this increase in insulin level, we studied normal mongrel dogs with 2DG alone, 2DG during ganglionic blockade, beta-adrenergic blockade, and postganglionic parasympathetic blockade. There was an increase in plasma epinephrine, norepinephrine, pancreatic polypeptide, insulin, and glucose post-2DG alone. During ganglionic blockade, the increase in epinephrine, norepinephrine, and pancreatic polypeptide post-2DG was completely abolished, verifying ganglionic blockade of sympathetic and parasympathetic pathways, respectively. Despite this, ganglionic blockade failed to abolish the insulin rise after 2DG. Postganglionic parasympathetic blockade did not change the insulin rise after 2DG. However, beta-adrenergic blockade completely abolished the insulin rise after 2DG. The above data suggests that 1) the insulin rise post-2DG is beta-adrenergic but 2) the ganglionic neurotransmitter mediating the 2DG-induced insulin rise during ganglionic blockade is noncholinergic (possibly peptidergic).
Asunto(s)
Epinefrina/metabolismo , Ganglios/fisiología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Norepinefrina/metabolismo , Animales , Perros , Epinefrina/sangre , Hexametonio , Compuestos de Hexametonio/farmacología , Insulina/sangre , Secreción de Insulina , Islotes Pancreáticos/inervación , Cinética , Masculino , Norepinefrina/sangre , Polipéptido Pancreático/sangre , Polipéptido Pancreático/metabolismoRESUMEN
A sensitive assay was used to measure the binding of iodine-125-labeled insulin in serum obtained from 112 newly diagnosed insulin-dependent diabetics before insulin treatment was initiated. Two groups of nondiabetics served as controls: children with a variety of diseases other than diabetes and nondiabetic siblings of insulin-dependent diabetics. Eighteen of the diabetics were found to have elevated binding and 36 were above the 95th percentile of control values. The insulin-binding protein is precipitated by antibody to human immunoglobulin G, has a displacement curve that is parallel and over the same concentration range as serum from long-standing insulin-dependent diabetics, and elutes from a Sephacryl S-300 column at the position of gamma globulin. These insulin antibodies are present in a large percentage of newly diagnosed, untreated diabetics and may be an immune marker of B-cell damage.
Asunto(s)
Autoanticuerpos/análisis , Diabetes Mellitus Tipo 1/inmunología , Insulina/inmunología , Adolescente , Adulto , Niño , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Humanos , Insulina/uso terapéuticoRESUMEN
The amounts and concentrations of secretin in extracts of rat and guinea pig small intestine were measured with a porcine secretin radioimmunoassay. Immunoactivity in the extracts comigrated with porcine secretin in sodium dodecyl sulfate acrylamide gel electrophoresis. In the small intestines of adult rats and newborn and adult guinea pigs, there was a marked proximal-to-distal gradient in secretin concentration; newborn rats exhibited approximately equal proximal and distal secretin concentrations. Concentrations were as follows: most proximal newborn or adult guinea pig segments, 80 ng/mg prot; most distal newborn or adult guinea pig segments, 2 ng/mg prot; most proximal adult rat segments, 50 ng/mg prot; most distal adult rat segments, 8 ng/mg prot; and newborn rat intestine, 11 ng/mg prot. In either rats or guinea pigs, the secretin concentration in the proximal small intestine fell between days 2 and 6 postpartum to levels 3- and 10-fold below their respective newborn values. It is consistent with the relatively mature state of the newborn guinea pig digestive tract that it displays the proximal-to-distal gradient of secretin that is characteristic of both adult guinea pigs and rats. In the proximal intestine of both species, intestinal growth outpaces the accumulation of secretin.
Asunto(s)
Intestino Delgado/crecimiento & desarrollo , Secretina/metabolismo , Envejecimiento , Animales , Animales Recién Nacidos , Cobayas , Especificidad de Órganos , Radioinmunoensayo/métodos , Ratas , Especificidad de la EspecieRESUMEN
Three forms of immunoreactive pancreatic polypeptide (PPI) were detected in extracts of cultured dog pancreatic PP cells: PPI of (1) larger apparent molecular weight than PP, (2) similar apparent molecular weight but different isoelectric point than PP, and (3) identical apparent molecular weight and isoelectric point with PP. Dog pancreatic endocrine cells in culture were labeled biosynthetically with tritiated amino acids, and extracted proteins were fractionated by sodium dodecyl sulfate gel electrophoresis. A total of 97% of the PPI migrated like PP itself while about 3% of the PPI migrated like proteins up to 7200 molecular weight. PPI migrating like PP was analyzed further by isoelectric focusing and was found to occur in a neutral form like PP and a more acidic form. Peptide mapping of neutral and acidic PPI forms showed that both were like PP with the exception that the C-terminal [3H]tyrosine-containing peptide was a peptide with a net negative charge of 1 arising from a peptide extension of one or a few amino acids. The acidic form of PP was also shown to occur in pancreas extracts. However, neutral PPI was 90% of the total PPI in the pancreas extracts while the converse was true of culture extracts. We conclude that culturing the PP cell affects the efficiency of the process of amidation, that acidic PP could be either biosynthetic precursor or end product, and that the existence of the larger PP form(s) signals (signal) the possible production of yet other peptides by the PP cell.