Cellular localization of the inhibitory action of relaxin against uterine spasm.

1. The aim of this study was to determine whether the site of action of relaxin as a relaxant of rat myometrium is at the cell membrane or at an intracellular-site. Therefore, the potency of relaxin was determined against spasms reliant predominantly upon either extracellular Ca2+ or intracellular Ca2+. Uterine spasms dependent upon extracellular Ca2+ were elicited by (i) oxytocin (0.2 nM) (ii) Bay K 8644 (1 microM) in 10 mM K(+)-rich PSS and (iii) KCl (80 mM). Uterine spasm dependent upon intracellular Ca2+ was elicited by oxytocin (20 nM) in the presence of nifedipine (500 nM). The effects of relaxin against these spasmogens were compared with those of levcromakalim, nifedipine and salbutamol. 2. Relaxin (0.2-6.3 nM), levcromakalim (25-800 nM), salbutamol (1-63 nM) and nifedipine (1-250 nM) caused concentration-dependent inhibition of the spasm evoked by oxytocin (0.2 nM) and relaxin was the most potent relaxant. 3. Relaxin and nifedipine were slightly less potent against the spasm induced by Bay K 8644 (1 microM) than against spasm induced by oxytocin (0.2 nM) (15 fold and 13 fold respectively). Levcromakalim and salbutamol were equipotent against the spasm evoked by Bay K 8644 (1 microM) and that evoked by oxytocin (0.2 nM). 4. Relaxin induced only 47 +/- 7% inhibition of the KCl (80 mM)-evoked spasm at a concentration of 0.8 microM. Levcromakalim was much less potent (427 fold) against the spasm evoked by KCl (80 mM) than against the spasm evoked by oxytocin (0.2 nM). The potency of salbutamol against the spasm evoked by KCl (80 mM) was modestly reduced (14 fold) compared to that against the spasm evoked by oxytocin (0.2 nM). The potency of nifedipine against the KCl (80 mM)-evoked spasm was not different from that against the oxytocin (0.2 nM)-evoked spasm. 5. The potencies of relaxin and levcromakalim against the spasm evoked by oxytocin (20 nM) + nifedipine (500 nM) were greatly reduced (74 fold and 234 fold respectively) compared to their potencies against the spasm evoked by oxytocin (0.2 nM). The potency of salbutamol against these two spasmogens was not different. 6. Relaxin was much less potent against the spasm dependent upon intracellular Ca2+ (that induced by oxytocin (20 nM) + nifedipine (500 nM)) than against the spasms dependent upon extracellular Ca2+, those induced by oxytocin (0.2 nM) and Bay K 8644 (1 microM). In this regard, relaxin resembled levcromakalim and nifedipine rather than salbutamol. Therefore, the major site of action of relaxin appears to be located at the plasma membrane rather than at an intracellular level. The observation that relaxin was less effective against the KCl (80 mM)-induced spasm than against the oxytocin (0.2 nM)-evoked spasm may indicate that relaxin has a minor action involving K(+)-channel opening. 7. High concentrations of relaxin (up to 1 microM) induced significant inhibition of the spasm dependent upon intracellular Ca2+. Thus at high concentrations relaxin also appears to have an additional intracellular action.     

Posttraumatic cerebral arterial spasm: transcranial Doppler ultrasound, cerebral blood flow, and angiographic findings.

Thirty patients admitted after suffering closed head injuries, with Glasgow Coma Scale scores ranging from 3 to 15, were evaluated with transcranial Doppler ultrasound monitoring. Blood flow velocity was determined in the middle cerebral artery (MCA) and the intracranial portion of the internal carotid artery (ICA) in all patients. Because proximal flow in the extracranial ICA declines in velocity when arterial narrowing becomes hemodynamically significant, the extracranial ICA velocity was concurrently monitored in 19 patients. To assess cerebral perfusion, cerebral blood flow (CBF) measurements obtained with the intravenous 133Xe technique were completed in 16 patients. Vasospasm, designated as MCA velocity exceeding 120 cm/sec, was found in eight patients (26.7%). Severe vasospasm, defined as MCA velocity greater than 200 cm/sec, occurred in three patients, and was confirmed by angiography in all three. Subarachnoid hemorrhage (SAH) was documented by computerized tomography in five (62.5%) of the eight patients with vasospasm. All cases of severe vasospasm were associated with subarachnoid blood. The time course of vasospasm in patients with traumatic SAH was similar to that found in patients with aneurysmal SAH; in contrast, arterial spasm not associated with SAH demonstrated an uncharacteristically short duration (mean 1.25 days), suggesting that this may be a different type of spasm. A significant correlation (p less than 0.05) was identified between the lowest CBF and highest MCA velocity in patients during the period of vasospasm, indicating that arterial narrowing can lead to impaired CBF. Ischemic brain damage was found in one patient who had evidence of cerebral infarction in the territories supplied by the arteries affected by spasm. These findings demonstrate that delayed cerebral arterial spasm is a frequent complication of closed head injury and that the severity of spasm is, in some cases, comparable to that seen in aneurysmal SAH. This experience suggests that vasospasm is an important secondary posttraumatic insult that is potentially treatable.     

Giant cavernous hemangiomas: Diagnosis and surgical strategies

From January, 1972 to June, 1989, 51 patients with liver hemangiomas (32 females and 19 males, mean age 35 years) were evaluated for surgical treatment. Diameters of the masses were 5 cm to 20 cm (median 8.5 cm). Nine of the patients had already been treated for cancer. Twenty-two (43.1%) of the 51 patients were symptomatic and 29 (56.9%) patients were asymptomatic. In 34 patients (66.7%) a definite diagnosis of hemangioma was made by scintiscan and/or ultrasound and/or computed tomography and/or angiography while in the remaining 17 (33.3%) patients the diagnosis was uncertain. The most common indications for resection were the presence of a symptomatic angioma, a symptomatic mass with an uncertain diagnosis, and/or lack of a definite pre-operative diagnosis. Surgery was performed on 25 patients. Ten anatomic and 15 atypical resections or enucleations were performed. There were no postoperative deaths. Two further patients, operated for probable hemangioma, were found to have primary hepatic malignancies. In the 26 unresected patients, no complications were observed during follow-up. In 3 patients, hemangioma enlargement was detected by ultrasound, but there were no symptoms. As cavernous liver hemangiomas are now more reliably diagnosed and their natural history is usually uneventful, surgery can be avoided in most cases. However, when a non-resection policy is adopted, an exact diagnosis is essential in order to rule out primary or metastatic cancer. Surgical exploration and treatment should be limited to symptomatic or complicated cases as well as to patients with an uncertain diagnosis.

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Resumen Cincuenta y un pacientes con hemangiomas del higado (32 mujeres y 19 hombres, edad promedio 35 años) fueron valorados en cuanto a tratamiento quirúrgico en el período enero 1972 a junio de 1989; el diámetro de las lesiones oscilo entre 5 y 20 cm (promedio 8.5 cm). Nueve de los pacientes ya habían sido tratados por cáncer; 22/51 (43.1%) estaban sintomáticos y 29/51 (56.9%) eran asintomáticos. En 34/51 (66.7%) se hizo el diagnóstico definitivo de hemangioma mediante escintigrafia y/o ultrasonido y/o tomografía computadorizada y/o angiografia, en tanto que en los otros 17 pacientes (33.3%) el diagnóstico resultó incierto. Las indicaciones más comunes para resección fueron: presencia de un angioma sintomático, una masa asintomática con diagnóstico incierto y/o ausencia de diagnóstico preoperatorio definitivo. Se practicó cirugía en 25/51 pacientes, habiéndose realizado 10 resecciones anatómicas y 15 resecciones atípicas o enucleaciones. No hubo muertes postoperatorias. Otros dos pacientes operados por probable hemangioma demostraron tener neoplasias malignas hepáticas primarias. En los 26 pacientes no resecados no se observaron complicaciones durante el seguimiento; en tres casos se detectó ensanche del hemangioma en el examen con ultrasonido, pero no se presentaron síntomas. Puesto que actualmente los hemangiomas cavernosos del hígado pueden ser diagnosticados con mayor certeza y puesto que su historia natural generalmente está libre de complicaciones es posible evitar la cirugía en la mayoría de los casos. Sin embargo, cuando se pretenda adoptar una política de no resección es esencial establecer el diagnóstico exacto para excluir la posibilidad de un cáncer primario o metastásico. La exploración y el tratamiento quirúrgicos debe permanecer limitados a los hemangiomas sintomáticos o complicados, y en ningún caso deben significar riesgo para el paciente.

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Résumé De Janvier 1972 à Juin 1989, 51 patients ayant un hémangiome du foie (32 femmes et 19 hommes, âge moyen 35 ans) ont été examinés en vue d'une exérèse chirurgicale. Le diamètre de la lésion variait entre 5 et 20 cm (médiane = 8.5 cm). Neuf des patients avaient déjà été traités pour un cancer; 22/51 (43.1%) étaient symptomatiques alors que 29/51 (56.9%) étaient asymptomatiques. Chez 34/51 patients (66.7%), le diagnostic définitif d'hémangiome a été confirmé par scintigraphie et/ou échographie et/ou tomodensitométrie, alors que chez les 17 autres (33.3%), le diagnostic était uncertain. Les indications d'une résection les plus fréquentes étaient: la présence d'un angiome symptomatique, une masse symptomatique avec un diagnostic uncertain, et/ou absence de diagnostic définitif préopératoire. Vingt-cinq des 51 patients ont été opérés. Il y a eu 10 résections anatomiques et 15 résections atypiques ou énucléations. Il n'y a pas eu de mortalité postopératoire. Deux autres patients, traités pour ce que l'on soupçonnait être une probalbe hémangiome du foie, avaient en fait un cancer hépatique. Chez les 26 patients non résequés, il n'y avait pas eu de complication. Chez trois patients, on a mis en évidence une augmentation de volume par l'échographie, mais ces modifications ne s'accompagnait d'aucune symptomatologie. Comme on peut faire le diagnostic d'hémangiome carverneux du foie avec plus de fiabilité qu'avant, et comme on sait que leur histoire naturelle est généralement bénigne, on peut le plus souvent surseoir à l'exérèse chirurgicale. Cependant, il importe de toujours faire le diagnositc avec certitude, de façon à éliminer un cancer primitif ou sécondaire du foie.

     

Excitatory amino acid receptor regulation after subthalamic nucleus lesions in the rat

The subthalamic nucleus plays a pivotal role in the regulation of basal ganglia output. Recent electrophysiologic, lesion and immunocytochemical studies suggest that the subthalamic nucleus uses an excitatory amino acid as a neurotransmitter. After complete ablation of the subthalamic nucleus, we have examined the NMDA, AMPA, kainate and metabotropic subtypes of excitatory amino acid receptors in two major subthalamic projection areas (globus pallidus and substantia nigra pars reticulata) with quantitative autoradiography. Two weeks after ablation, binding sites for [³H]AMPA and [³H]kainate increased in substantia nigra pars reticulata ipsilateral to the lesion. In globus pallidus on the lesioned side, [³H]glutamate binding to the NMDA recognition site decreased. The results suggest that glutamate receptors regulate after interruption of subthalamic nucleus output.     

Diltiazem pharmacokinetics in the rat and relationship between its serum concentration and uterine and cardiovascular effects.

1 The kinetics of diltiazem were investigated in ovariectomized (ovx) non-pregnant and intact late pregnant anaesthetized rats following a bolus i.v. injection (2 mg kg-1) and during a 180 min i.v. infusion (50 micrograms kg-1 min-1 and 100 micrograms kg-1 min-1). Uterine contractions, mean blood pressure and heart rate were measured in the non-pregnant rats. 2 Measurement of serum diltiazem concentrations after bolus i.v. injection in ovx non-pregnant rats showed a biexponential decay with time from which the following parameters were calculated: volume of distribution area (V(area)) - 256 +/- 46 ml; rate constants k12 - 0.46 +/- 0.10 min-1; k21 - 0.09 +/- 0.01 min-1; kel - 0.13 +/- 0.03 min-1; elimination clearance - 3.2 +/- 0.3 ml min-1; distribution t1/2 (t1/2) - 1.4 +/- 0.3 min; elimination t1/2 (t1/2 beta) - 61.2 +/- 13.0 min. In pregnant rats, a biexponential decay was also observed with similar parameters to those in non-pregnant animals except for markedly increased V(area) - 1004 +/- 184 ml; kel - 0.54 +/- 0.16 min-1 and elimination clearance - 14.8 +/- 2.3 ml min-1. 3 Measurement of serum diltiazem concentrations during infusion yielded the following parameters in non-pregnant ovx rats: V(ss)--79 +/- 10 ml; rate constants k12 - 1.02 +/- 0.21 min-1; k21 - 0.03 +/- 0.01 min-1; kel - 0.39 +/- 0.06 min-1; elimination clearance - 7.8 +/- 1.2 ml min-1. In pregnant rats a marked increase was observed in kel - 1.25 +/- 0.38 min-1 and elimination clearance - 36.4 +/- 13.8 ml min-1. 4 An immediate reduction in uterine contractions, mean blood pressure and heart rate was observed after bolus i.v. injection of diltiazem with a return towards control values as serum diltiazem concentrations declined. There were significant correlations between the inhibition of the 3 parameters and the log serum concentrations of diltiazem. Serum concentration-response curves indicated IC50 values of 0.5 microgram ml-1 for inhibition of uterine contractions, 0.7 microgram ml-1 for reduction in blood pressure and 1.2 micrograms ml-1 for reduction in heart rate. There were maintained reductions in the integral of uterine contractions, mean blood pressure and heart rate during infusion. 5 The metabolite desacetyldiltiazem was rarely detected after i.v. bolus injection and was not found in 5/13 rats infused with diltiazem, yet significant inhibition of uterine contractions was observed in all rats. Diltiazem was 3.2 fold more potent than desacetyldiltiazem as an inhibitor of contractions of the rat isolated uterus.(ABSTRACT TRUNCATED AT 400 WORDS)     

MUC4 expression increases progressively in pancreatic intraepithelial neoplasia

Pancreatic adenocarcinoma is believed to develop from histologically identifiable intraductal lesions known as pancreatic intraepithelial neoplasias (PanINs) that undergo a series of architectural, cytologic, and genetic changes, a progression model similar to the adenoma-carcinoma sequence in the colon. The apomucin MUC4 has been implicated in invasive pancreatic adenocarcinoma. MUC4 expression is not detectable at the RNA level in normal pancreas but is detectable at high levels in invasive pancreatic adenocarcinoma. We documented the pattern of expression of MUC4 in PanINs by studying a series of 71 PanIN lesions immunohistochemically using a new monoclonal antibody to MUC4. Five (17%) of 30 PanIN-1 lesions, 10 (36%) of 28 PanIN-2 lesions, 11 (85%) of 13 PanIN-3 lesions, and 25 (89%) of 28 invasive adenocarcinomas labeled with the MUC4 antibody used in the study. In addition, afew nonneoplastic lesions labeled with the MUC4 antibody, including reactive ducts in chronic pancreatitis, atrophic ducts filled with inspissated secretions, and ducts showing squamous metaplasia. Our data help establish the patterns of MUC4 expression in neoplastic precursors in the pancreas and add further support to the progression model for pancreatic adenocarcinoma.     

Influence of organ site and tumor cell type on MUC1-specific tumor immunity

We investigated the influence of organ-specific parameters on tolerance and immunity to human MUC1. C57Bl/6 mice (wild-type) and C57Bl/6 transgenic for MUC1 (MUC1.Tg) were challenged in the pancreas with Panc02-MUC1, a C57Bl/6-syngeneic pancreatic cancer cell line expressing human MUC1. Wild-type mice produced immune responses to MUC1 when presented on tumor cells growing in the pancreas; however, the responses to tumors in the pancreas were less effective than responses produced by tumor challenge at the s.c. site. Tumor immunity specific for MUC1 was produced in wild-type mice by two different procedures: (i) s.c. immunization of wild-type mice with a low dose of Panc02-MUC1 or (ii) adoptive transfer of spleen and lymph node cells harvested from wild-type mice previously immunized s.c. with Panc02-MUC1. This demonstrates that immune responses to MUC1 presented at the s.c. site can be detected and adoptively transferred. MUC1.Tg mice were immunologically tolerant to MUC1; however, some immunological protection against orthotopic challenge with Panc02-MUC1 was conferred by adoptive transfer of CD4+ and CD8+ T cells from wild-type mice. These results show that it is more difficult to produce immune responses to tumors growing at the pancreatic site than the s.c. site. Panc02-MUC1 cells growing in the pancreas were accessible to the immune system, and immune responses evoked by s.c. presentation of this molecule in wild-type mice were effective in rejecting tumor cells in the pancreas of both wild-type and MUC1.Tg mice. No effective anti-tumor immune responses against MUC1 were produced in MUC1.Tg mice.     

Characterization of the structural elements in lipid A required for binding of a recombinant fragment of bactericidal/permeability-increasing protein rBPI23.

Both human bactericidal/permeability-increasing protein (BPI) and a recombinant amino-terminal fragment of BPI (rBPI23) have been shown to bind with high affinity to the lipid A region of lipopolysaccharide (LPS) (H. Gazzano-Santoro, J. B. Parent, L. Grinna, A. Horwitz, T. Parsons, G. Theofan, P. Elsbach, J. Weiss, and P. J. Conlon, Infect. Immun. 60:4754-4761, 1992). In the present study, lipid A preparations derived from bacterial LPS as well as synthetic lipid A's and various lipid A analogs were used to determine the structural elements required for rBPI23 binding. rBPI23 bound in vitro to a variety of synthetic and natural lipid A preparations (both mono- and diphosphoryl forms), including lipid A's prepared from Escherichia coli and Salmonella, Neisseria, and Rhizobium species. Binding does not require that the origin of negative charge be phosphate, since rBPI23 bound with high affinity to lipid A's isolated from Rhizobium species that contain carboxylate (Rhizobium trifolii) or sulfate (Rhizobium meliloti) anionic groups and lack phosphate. Lipid A acyl chains are important, since rBPI23 did not bind to four synthetic variants of the beta(1-6)-linked D-glucosamine disaccharide lipid A head group, all devoid of acyl chains. rBPI23 also bound weakly to lipid X, a monosaccharide lipid precursor of LPS corresponding to the reducing half of lipid A. Lipid IVA, a precursor identical to E. coli lipid A except that it lacks the 2' and 3' acyl chains, was the simplest structure identified in this study that rBPI23 bound with high affinity. These results demonstrate that rBPI23 has a binding specificity for the lipid A region of LPS and binding involves both electrostatic and hydrophobic components.