Emergencies in general practice.
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Emergencies↗
Biomedical subjects
C W Cooper
Publications and source records attributed to C W Cooper.
Effect of transforming growth factor beta on parathyroid hormone receptor binding and cAMP formation in rat osteosarcoma cells.
Transforming growth factor beta (TGF-beta) is now recognized as an important growth regulator and modulator in bone, where it apparently acts in an autocrine or paracrine fashion. In an effort to help elucidate how TGF-beta may interact with parathyroid hormone (PTH) to influence bone turnover, we examined the idea that TGF-beta might alter the number or affinity of PTH receptors in osteoblastic bone cells, PTH receptor binding was assessed in cultured ROS 17/2.8 cells using [125I]PTHrP-(1-34) as labeled ligand. Specific binding to intact cells was measured in the presence of up to 1 microM unlabeled rPTH-(1-34), and cAMP in cell extracts was determined by RIA. Incubation of ROS cells with 2 ng/ml of TGF-beta for the maximally effective time of 3 days increased the number of PTH binding sites (Bmax) by 47 +/- 13%, with no change in the KD (3 nM). TGF-beta also increased the intracellular cAMP response to 0.3 nM rPTH-(1-34) (ED50) by 53 +/- 22%. Both effects were dose dependent, with 1-4 ng/ml of TGF-beta producing maximal effects, and both effects were blocked by the protein synthesis inhibitor cycloheximide (2-5 microM). Since TGF-beta induced comparable increases in both PTH binding and cAMP formation, the findings suggest that TGF-beta can increase the number of functional PTH receptors in cultured ROS 17/2.8 cells. This effect may reflect an action of TGF-beta to slow replication and promote differentiated functions in these cells.
Animals↗
Medical students' perceptions of an undergraduate general practice preceptorship.
A survey was undertaken to evaluate an undergraduate general practice preceptorship by means of an open-ended six-item questionnaire given to students at the end of their general practice attachment. The aim of the survey was to evaluate the preceptorship as a learning experience and to examine ways in which the preceptorship might be improved. Questionnaires from two medical student years (1985 and 1990) were examined retrospectively. Of 398 potential responders, 386 (97%) had completed questionnaires. Students expressed a very high level of satisfaction with the attachment and this was expressed most often in terms of the personal interest shown by the general practitioner, the variety of clinical problems encountered, and the experience gained in managing common clinical problems. Areas of the attachment which students felt could be improved were: involvement in the consultation, the time constraints on teaching and the teaching of practical skills. The paper discusses possible improvements which could be made to preceptorship programmes on the basis of these findings.
Education, Medical↗
Effects of parathyroid hormone-related peptide on adenosine 3',5'-monophosphate and ornithine decarboxylase in a human colonic cell line.
PTH-related peptide (PTHrP) is widely distributed in normal tissues, including the gut, and is considered a potential autocrine or paracrine regulator of cellular growth and differentiation. With this in mind, a human colonic cell line (LoVo) was used to study the effect of PTHrP on ornithine decarboxylase (ODC), because ODC is known to have profound effects on the growth and differentiation of many cell types via stimulation of synthesis of polyamines. cAMP also was measured, because this second messenger has been implicated in the regulation of ODC activity. Nearly confluent LoVo cells, grown in F-12 medium and 10% fetal bovine serum (FBS), were preincubated in 1% FBS for at least 5 h, and then PTHrP-(1-34) was added, and the incubation was continued for up to 6 h. Cell extracts were analyzed for ODC activity by measuring 14CO2 liberated from 14C-labeled ornithine, for cAMP by RIA, and for ODC mRNA by Northern analysis. PTHrP produced dose-related increases in both cAMP (2- to 3-fold) and ODC (3- to 5-fold), with a maximal effect at 0.1-1 microM and an ED50 of 1-10 nM. Comparison of the cAMP and ODC responses to PTHrP showed a strong correlation (r = 0.96; P less than 0.001). The effects of 1 microM PTHrP-(1-34) to increase cAMP and ODC were completely inhibited by 10-20 microM of the specific antagonist [Asn10,Leu11]PTHrP-(7-34). PTHrP-(1-34) did not stimulate ODC activity when cells were incubated without FBS. The stimulation of ODC activity by PTHrP-(1-34) was maximal at 2 h, a time at which an increase in ODC mRNA also was evident. PTH-(1-34) and forskolin also stimulated ODC activity, but PTHrP-(67-86) amide was ineffective. The results indicate that the N-terminal portion of the PTHrP molecule can stimulate ODC activity in a human colon cell line and that the effect is probably mediated by cAMP. The results are consistent with the idea that PTHrP may influence cell growth and differentiation in the gut via an effect on polyamine biosynthesis. Since LoVo cells also express PTHrP mRNA, this gastrointestinal cell line may serve as a useful model for studying autocrine regulation of gut cell growth and differentiation by PTHrP.
Cell Line↗
Effects of vasoactive intestinal peptide on adenosine 3',5'-monophosphate, ornithine decarboxylase, and cell growth in a human colon cell line.
Vasoactive intestinal peptide (VIP) is a widely distributed neuropeptide that has been considered a potential regulator of cell growth and differentiation in various tissues, including the gut. To examine this idea, we used a human colon carcinoma cell line (LoVo) as a model system and measured ornithine decarboxylase (ODC), because this is the rate-limiting enzyme for the formation of polyamines, which are thought to be key factors in regulating cell growth. LoVo cells, grown to about 80% confluence in F-12 medium containing 10% fetal bovine serum, were preincubated for 5 h in low serum medium (1% fetal bovine serum in F-12), and ODC activity was determined by measuring 14CO2 liberated from 14C-labeled ornithine. VIP caused a dose-related biphasic change in ODC, with activity increased at 10 pM, maximal (5-fold increase) at 10 nM, and decreased toward basal at 100 nM to 1 microM. Incubation of cells for 6 days with VIP in low serum medium showed similar changes in cell numbers, with growth being increased by doses in the 1 pM to 100 nM range and decreased at higher doses (greater than or equal to 100 nM). Exposure of cells to 5 mM alpha-difluoromethylornithine blocked both the VIP-induced increase in cell number and the VIP-induced increase in ODC activity. Increased ODC mRNA was detected after 2 h of exposure to VIP, a time at which ODC activity peaked after treatment, and the increase in ODC mRNA caused by VIP was dose-dependent. In related experiments LoVo cells were found to have high affinity VIP receptors (Kd = 0.4 nM), as assessed by examination of [125I]VIP binding in the presence of varying concentrations of unlabeled VIP. Studies of intracellular cAMP revealed a dose-related increase in cAMP in response to VIP (ED50 = 11 pM), and the adenylate cyclase activator forskolin increased both ODC activity and ODC mRNA. The findings support the idea that LoVo cells have VIP receptors linked to cAMP which can stimulate cell growth at least in part by increasing ODC synthesis and activity, thereby altering the production of polyamines. The decreased growth and ODC activity observed with high doses of VIP may involve a second messenger other than cAMP.
Adenocarcinoma↗
Role of extracellular magnesium in insulin secretion from rat insulinoma cells.
Magnesium (Mg2+) is an abundant intracellular cation that participates in the regulation of the intracellular concentration of ATP. In this study, we examined the relationship between insulin secretion and intracellular free Mg2+ ([Mg2+]i) in a rat-insulinoma cell line (RIN m5F), using a fluorescent dye (Mag-fura-2). KCI, forskolin, and D-glyceraldehyde increased [Mg2+]i and insulin secretion from RIN m5F cells in a dose-dependent fashion. Verapamil, a voltage-dependent Ca2+ channel blocker, inhibited the increase of [Mg2+]i that was evoked by KCI, forskolin, and D-glyceraldehyde. In a Mg(2+)-free buffer, these agents failed to cause an elevation in [Mg2+]i; however, the insulin response to KCI and forskolin was enhanced, compared with that in the presence of Mg2+ (1.25 mM). Our findings suggest that [Mg2+]i is dependent upon extracellular Mg2+, and the influx through the voltage-dependent Ca2+ channel. Mg2+ may competitively inhibit the voltage-dependent Ca2+ channel, which is known to play a role in insulin secretion. An absence of Mg2+ in the extracellular space may result in enhanced insulin secretion. [Mg2+]i may play a role in insulin secretion from RIN m5F cells.
Animals↗
Prevalence of antibody to Brucella in asymptomatic well individuals in Saudi Arabia.
Two populations of asymptomatic well individuals resident in central Saudi Arabia were tested for antibody to brucella antigen. A relatively high prevalence of antibody (as measured by the standard agglutination test) was found in both populations. A much lower prevalence of antibody was demonstrated by the 2-mercaptoethanol test in these populations. There follows a discussion of the utility of brucella serological tests in developing countries in which the disease is endemic.
Adolescent↗
Expression of cholecystokinin forms by medullary thyroid cancer.
We have studied the production and release of cholecystokinin (CCK) forms by rat medullary thyroid cancer (MTC) in vivo. MTC cells were inoculated s.c. into 8 Wag-Rij rats. One month later, after i.v. injection of calcium plasma levels of CCK, calcitonin and tissue contents of gastrin and calcitonin were determined by radioimmunoassay (RIA), immunocytochemistry, and high-pressure liquid chromatography (HPLC). The tumors were passed 4 times to 8 rats in 1-month intervals fasting levels of CCK in control rats were unaffected by calcium stimulation, and in tumor-bearing rats, plasma CCK was elevated in 3 out of 4 passages, falling to normal levels at the end of passage 4. Hypercalcemia had no effect on plasma levels of CCK in tumor-bearing rats, but did stimulate the release of calcitonin in both control and some tumor-bearing rats in later passages. CCK-8 sulfate was found in all 4 tumor passages but not CCK-33/39. We conclude that rat MTC synthesizes and releases CCK-8, but unlike calcitonin, release of CCK appears unresponsive to calcium stimulation.
Animals↗
Stimulation of calcitonin secretion in the pig by calcitonin gene-related peptide.
Pig thyroid glands were surgically isolated in situ and perfused with autologous blood to which was added known concentrations of calcitonin gene-related peptide (alpha CGRP). When thyroids were perfused with measured concentrations of CGRP within the range of 0.6-600 nM, the secretion rate of calcitonin (CT) was stimulated while the release of T3, T4, and somatostatin remained unchanged. Specific binding of 125I-CGRP to pig thyroid plasma membranes was demonstrated, and binding was inhibited by unlabelled CGRP but not by CT or by other peptides unrelated structurally to CGRP. The findings indicate that the pig thyroid gland contains plasma membrane binding sites for CGRP and that CGRP is capable of stimulating the secretion of CT.
Animals↗
Effects of parathyroid hormonal peptides on the gut.
Our previous studies have shown PTH to be an effective relaxant of smooth muscle throughout the mammalian tract. Recently, we found PTHrP to be equally as potent and effective on the gut as PTH, and we hypothesized that PTHrP, rather than PTH, might be the natural ligand for the gut receptors which mediate GI smooth muscle relaxation. To approach this question, we asked whether rat GI tissue expresses mRNA for PTHrP. Using selective reverse transcription and PCR we have found PTHrP mRNA in smooth muscle throughout the rat GI tract and in gastric and colonic mucosa as well. Our findings support the idea that PTHrP can be produced by GI tissues and that it may function there as an autocrine or paracrine factor. One of its actions may involve regulation of GI muscle tone.
Animals↗
The epidemiology of human brucellosis in a well defined urban population in Saudi Arabia.
A retrospective survey was undertaken to provide the first reported estimate of the incidence of human brucellosis in Saudi Arabia. The study population was unusually well defined and consisted of all individuals resident in the Riyadh and Al Kharj regions and registered for treatment in the Riyadh-Al Kharj Hospital Program. Cases satisfying predetermined case criteria were identified initially from a retrospective review of hospital laboratory records, and this was supported by a review of individual medical case-notes. Brucellosis was found to be much more common in Saudi nationals than expatriate nationals. Among Saudi nationals the study demonstrated a remarkable increase in brucellosis with increasing age, and a higher incidence amongst women than men in some age groups. This was felt to have been due either to an increased exposure to infected livestock, or to an increased susceptibility to the disease in women, and with increasing age. There was a seasonal fluctuation in the occurrence of brucellosis with the largest number of cases occurring in spring and summer.
Adolescent↗
Radioiodinated rat parathyroid hormone-(1-34) binds to its receptor on rat osteosarcoma cells in a manner consistent with two classes of binding sites.
Binding of 125I-labeled rat (r) PTH-(1-34) to ROS 17/2.8 osteoblastic bone cells and to membranes from these cells was examined. Competitive binding inhibition experiments were performed using unlabeled rPTH-(1-34) with particular emphasis on concentrations of peptide below 1 nM. In intact cells, binding of labeled rPTH-(1-34) was highly specific, and inhibition of binding by unlabeled ligand suggested the presence of two classes of binding sites, one with high affinity and low capacity (KD = 40 pM, approximately 20% of total binding sites) and the other with lower affinity and high capacity (KD = 2 nM, approximately 80% of the sites). Membranes prepared from ROS cells also exhibited a pattern of binding from competitive inhibition curves consistent with two distinct binding sites (KD = 30 pM and 6 nM). In intact ROS cells, cellular cAMP levels increased over the range of 10(-11)-10(-9) M rPTH-(1-34) with an ED50 intermediate between the two KD values (0.25 nM). These data suggest that osteoblastic bone cells possess two distinct classes of membrane receptors for PTH. Since the KD of the higher affinity site more closely approximates circulating concentrations of PTH, binding to this site may have physiologic relevance.
Adenylyl Cyclases↗
Skeletal effects of sodium fluoride during hypokinesia.
This study tested the capacity of fluoride (F) to prevent the disuse-associated reduction in bone formation/growth. Suspending young male Wistar rats by the tail for 2-2.5 weeks reduced femoral cortical (P less than 0.05) and trabecular (P less than 0.01) bone areas. Tetracycline labelling showed that the decrement in cortical area was largely due to a reduction in the percent periosteal mineralizing surface (PsMS). Periosteal mineral apposition rate (PsMAR) was not affected. Endosteal mineralizing surface (EsMS) and mineral apposition rate (EsMAR) were significantly stimulated spontaneously during the second week of suspension. F treatment (5 mg/kg/day i.p.) prevented the loss in bone area, and established a trend toward increased PsMS without affecting EsMS and EsMAR. None of these changes are associated with alterations in serum Ca, P or osteocalcin. F treatment in hypokinetic animals caused a decrease in serum PTH (-21% compared to control; P = 0.001). We conclude that F prevents the development of hypokinetic osteopenia in rats.
Animals↗
Effect of dietary calcium supplementation on blood pressure and calciotropic hormones in mineralocorticoid-salt hypertension.
In order to determine the effect of dietary calcium supplementation on blood pressure and calciotropic hormones, we studied two groups (n = 12 each) of mineralocorticoid [deoxycorticosterone (DOC)]-salt hypertensive rats, one receiving a normal-calcium diet (0.6% calcium, as calcium carbonate) and the other a high-calcium diet (2.5% calcium), over an 8-week period. Dietary calcium supplementation significantly attenuated the rise in blood pressure. Serum ionized calcium concentrations were significantly decreased from baseline levels in both groups but tended to be higher among the calcium-supplemented rats. Serum concentrations of parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25-D) were significantly higher in the DOC-salt rats than in normotensive rats fed normal rat chow [PTH: 49 +/- 4 versus 15 +/- 0.9 pg/ml (means +/- s.e.m.); 1,25-D: 108 +/- 7 versus 73 +/- 13 pg/ml, in DOC-salt and normotensive rats, respectively]. In the DOC-salt rats, dietary calcium supplementation did not significantly lower the elevated serum concentration of PTH (from 49 +/- 4 to 40 +/- 4 pg/ml; NS), but did significantly reduce that of 1,25-D (from 108 +/- 7 to 66 +/- 8 pg/ml; P less than 0.01). Since 1,25-D may increase vascular smooth muscle calcium uptake, dietary calcium supplementation may lower blood pressure in DOC-salt hypertension, in part, by suppressing 1,25-D.
Animals↗
Cosecretion of calcitonin and calcitonin gene-related peptide from cultured rat medullary thyroid C cells.
Whether C cells cosecrete calcitonin (CT) and CGRP was examined by exposing cultured rat medullary thyroid carcinoma 6-23 cells for 2 h to high medium Ca and to agents with a potential for affecting Ca-dependent secretion. In every experiment exposure of cells to high medium Ca (2.0-2.5 mM) provoked an increased release of both peptides that was highly correlated (r = 0.73). With other test substances, also, changes in both hormones occurred in parallel. The Ca-channel activator, BAY-K-8644 (10 microM) increased secretion, and this was inhibited by the Ca channel blocker, nitrendipine (10 microM). The Ca2+ ionophore, ionomycin (5 microM), increased release, and the calmodulin-Ca channel inhibitor, phenytoin (100 microM), inhibited Ca-induced release. The active 4 beta isomer of phorbol-12,13-didecanoate (0.1 microM), but not the inactive 4 alpha isomer, increased secretion. The findings suggest that pathways mediating C cell secretion include plasma membrane Ca channels, intracellular [Ca2+], calmodulin, and protein kinase C. The results show that the secretory process in rat C cells involves the release of CGRP as well as CT.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Parathyroid hormone-related protein relaxes rat gastric smooth muscle and shows cross-desensitization with parathyroid hormone.
Previously, we reported that parathyroid hormone (PTH) is a potent and effective relaxant of rat gastrointestinal smooth muscle. Since the recently discovered PTH-related protein (PTHrP) has amino-terminal homology with PTH and acts like PTH on bone and kidney, we decided to study the effects of synthetic PTHrP analogs on the isometric tension of rat fundic strips. Rat (r) PTH-(1-34), human (h) PTHrP-(1-34), and [Tyr0]hPTHrP-(1-34) relaxed acetylcholine-stimulated fundic strips in a dose-dependent manner, with an IC50 of 6, 10, and 31 nM, respectively. However, maximal doses of [Tyr0]hPTHrP-(1-34) were considerably less effective than the other two peptides. Addition of rPTH-(1-34) or hPTHrP-(1-34) to a maximally effective dose of [Tyr0]hPTHrP-(1-34) produced no further relaxation, indicating that [Tyr0]hPTHrP-(1-34) also has antagonistic properties. Bovine PTH-(3-34) an established in vitro antagonist of PTH, partially inhibited the relaxant effect of PTHrP. Fundic strips that had been desensitized by preincubation for 30-45 minutes with either rPTH-(1-34) or hPTHrP-(1-34) (330-500 nM) were also insensitive to the relaxant action of either peptide, but in the same preparations, the relaxation produced by vasoactive intestinal peptide was unaffected. These studies indicate that PTHrP and PTH can interact with the same receptor. Whether PTHrP influences gastrointestinal motility in normal or tumor-bearing persons remains to be investigated.
Animals↗
Identification and characterization of parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding.
Parathyroid hormone (PTH) receptors have been described in renal tissue from several species, but not in the rat. In this study, radioligand binding techniques were used to identify and characterize PTH receptors in rat kidney cortical membranes. The sulfur-free PTH analog [Nle8,18Tyr34]bovine PTH-(1-34)amide was iodinated using the iodogen method. This ligand was suitable for use in identifying PTH receptors in canine renal membranes, but not rat renal membranes. Synthetic, unsubstituted rat PTH-(1-34) was iodinated using the milder, lactoperoxidase technique and was purified by HPLC on a C8 column. [125I]rat PTH-(1-34) bound rapidly to both rat and dog renal membranes. At 22 degrees C reaction reached steady state within 20 minutes, and this level was maintained for at least 3 h. Specific binding was routinely greater than 90% for rat kidney and greater than 95% for dog kidney. Similar results were obtained at 4 degrees C with a longer time required to attain steady state (approximately 45 minutes). Binding was reversible as demonstrated by dissociation of bound ligand after either infinite dilution or displacement with excess nonradioactive PTH. Binding was saturable and of high affinity (rat kidney: Bmax = 2.3 pmol/mg protein, Kd = 3.1 nM, dog kidney: Bmax = 2.1 pmol/mg protein, Kd = 3.7 nM). Rat renal cortical adenylate cyclase activity was stimulated by rat PTH in a dose-dependent manner with an EC50 of 4 nM, a value in good agreement with the binding data. This study demonstrates the feasibility of identifying and characterizing parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding techniques.
Adenylyl Cyclases↗
Calcitonin gene-related peptide in neural tissues: a phylogenetic study.
A heterologous radioimmunoassay using a rabbit antiserum raised against human calcitonin gene-related peptide (CGRP) was used to measure levels of immunoreactive CGRP (IR-CGRP) in the brain, pituitary, and spinal cord in species representing all classes of vertebrates from cyclostomes to mammals, except amphibians. All the brain extracts except those from the trout, goldfish, and iguana demonstrated the presence of IR-CGRP. Pituitary extracts from all animals, except the ratfish, goldfish and trout, contained IR-CGRP. CGRP was present in all classes of animals tested and seems to be highly conserved in the nervous system, where it may act as a neurotransmitter or neuromodulator.
Animals↗