[D-lactic acidosis in a child with short bowel syndrome]. / Acidose d-lactique chez un enfant présentant un syndrome de grêle court.

INTRODUCTION: d-lactic acidosis is a rare and severe complication of short bowel syndrome in children that may result from important ileal bacterial overgrowth by lactobacilli. Intestinal flora (Lactobacilli) is responsible for the production of d-lactic acid after fermentation of food carbohydrates. OBSERVATION: We report on the case of a 6-year-old child with a short bowel syndrome treated with both home enteral and parenteral nutrition. The patient suddenly presented with acute neurological symptoms including dysarthria and disorientation. Biological analysis revealed metabolic acidosis, increased plasma d-lactic acid assessed by organic acid chromatography analysis and a very important increase in expired hydrogen during glucose breath test. Lactobacillus fermentum (known to produce d and L isomers of lactic acid) was isolated in the gastric liquid and rectal swabs. Clinical and biological evolution was rapidly favourable after treatment with intravenous sodium bicarbonate, antibiotic therapy and interruption of enteral nutrition. CONCLUSION: d-lactic acidosis should be suspected when neurological symptoms occur in a child with short bowel syndrome. They can be prevented by treating intestinal bacterial overgrowth.

Efficacy of methotrexate in pediatric Crohn's disease: a French multicenter study.

BACKGROUND: Immunosuppressors play a major role in maintaining remission in Crohn's disease (CD). In patients who do not tolerate or escape therapy with azathioprine (AZA)/6-mercaptopurine, there is a marked need for other immunosuppressive drugs. The aim of the present study was to evaluate the efficacy and safety of methotrexate (MTX) in children with active CD. METHODS: In a retrospective multicenter (n = 3) study, the efficacy of MTX to induce complete remission or a clinical improvement was analyzed in 61 children with active CD. RESULTS: CD was diagnosed at a mean age of 11.1 +/- 2.3 years, and MTX was introduced 3.1 +/- 2.2 years after diagnosis. Indications to use MTX were a nonresponse to or relapse under AZA (n = 42) or AZA intolerance/toxicity (n = 19). MTX improved or induced complete remission in 49 patients (80%), of whom 18 (29.5%) relapsed after 13 +/- 10 months of treatment. Under MTX medication, complete remission was observed in 39%, 49%, and 45% at 3, 6, and 12 months, respectively. Follow-up over at least 24 months in 11 children confirmed a sustained remission on MTX monotherapy up to 40 months. Adverse reactions were observed in 14 patients (24%), requiring discontinuation of MTX in 6 children (10%) (liver enzyme elevation, n = 2; varicella-zoster, n = 1; nausea, n = 3). MTX allowed corticosteroid discontinuation in 36 patients. CONCLUSIONS: MTX improved the clinical course in most pediatric CD patients who escaped or did not tolerate AZA. Short-time toxicity of MTX resulted in drug discontinuation in <10%. These data point to a beneficial and safe use of MTX in the treatment of pediatric CD.

Identification of an additional gene belonging to the alpha 2 adrenergic receptor family in the human genome by PCR.

We here describe the cloning of an additional gene, called alpha 2-1.8, which is similar to the previously cloned human alpha 2-adrenergic receptor located on chromosome 4. The alpha 2-1.8 gene was identified by using the polymerase chain reaction with primers specific for sequences in transmembrane regions 2 and 5 of the previously isolated human alpha 2-C4 and alpha 2-C10 adrenoceptor genes, which are localized on chromosomes 4 and 10, respectively. The new gene was identified by amplifying the 1.8 kb size fractionated region of PstI restriction cut human genomic DNA. The previously cloned alpha 2-C10 and alpha 2-C4 genes were recovered at their expected locations, 0.96 and 5.9 kb, respectively. We have identified 387 bases of the new alpha 2-1.8 gene, and its sequence is identical to the previously described alpha 2-C4 gene, but it is distinct from the alpha 2-C10 and alpha 2-C2 genes. Our results demonstrate that the alpha 2-C4 adrenergic receptor exists in more than one copy in the human genome.

Delineation of three pharmacological subtypes of alpha 2-adrenoceptor in the rat kidney.

1. Simultaneous computer modelling of plain and ARC 239- and guanoxabenz-masked [3H]-RX821002 saturation curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239-masked guanoxabenz competition curves revealed that the drugs bound to three alpha 2-adrenoceptor subtypes in the rat kidney with grossly differing selectivities. These alpha 2-adrenoceptor subtypes were termed alpha 2 A, alpha 2B1 and alpha 2B2. The order of affinities for [3H]-RX821002 for the adrenoceptor sites was alpha 2A greater than alpha 2B1 greater than alpha 2B2, the KdS being 0.62 +/- 0.05, 2.52 +/- 0.11 and 6.74 +/- 1.21 nM, respectively. The order of affinities for ARC 239 was alpha 2B1 greater than alpha 2B2 much greater than alpha 2A with KdS 4.78 +/- 1.04, 28.8 +/- 4.1 and 1460 +/- 270 nM, respectively. For guanoxabenz the order of affinities was alpha 2A greater than alpha 2B1 much greater than alpha 2B2 with KdS 99.7 +/- 15.1, 508 +/- 135 and 25,400 +/- 2400 nM, respectively. 2. Binding constants for 14 compounds for the three rat kidney alpha 2-adrenoceptor subtypes were determined by the simultaneous computer modelling of plain and ARC 239- and guanoxabenz-masked drug competition curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239-masked guanoxabenz competition curves. Of the 14 compounds tested, oxymetazoline and guanfacine were found to bind with low affinities to both of the alpha 2B1- and alpha 2B2-adrenoceptor but with high affinity to the alpha 2A-adrenoceptor. 3. (-)-Adrenaline and (-)-noradrenaline showed dissimilar order of affinities for the three alpha2-adrenoceptors. For (-)-adrenaline the order of affinities was alpha2Bl >- alpha2A> alpha2B2 and for (-)-noradrenaline alpha2B2 > alpha2Bl > alpha2A. All three alpha2-adrenoceptors showed the expected stereoselective binding for adrenaline enantiomers, the (+)-form being 7-10 fold less potent than the (-)form. 4. [3H]-yohimbine was also used as radioligand. The data with this ligand were fully compatible with the [3H]-RX821002 data. However, [3H]-yohimbine appeared to label only alpha2Bl- and alpha2B2-adrenoceptors presumably because it had too low an affinity for alpha2A-adrenoceptors. 5. We conclude that three pharmacological subtypes of alpha2-adrenoceptors are labelled by [3H]-RX821002 in the rat kidney. Guanoxabenz and ARC 239 may be used in competition studies to delineate between these three alpha2-adrenoceptor subtypes. Moreoever, we here present a method allowing the determination of binding constants for an arbitrary drug to the three alpha2-adrenoceptor subtypes.

[3H]-MK 912 binding delineates two alpha 2-adrenoceptor subtypes in rat CNS one of which is identical with the cloned pA2d alpha 2-adrenoceptor.

1. Simultaneous computer modelling of control and guanfacine-masked [3H]-MK 912 saturation curves as well as guanfacine competition curves revealed that the drugs bound to two alpha 2-adrenoceptor subtypes in the rat cerebral cortex with very different selectivities. These alpha 2-adrenoceptor subtypes were designated alpha 2A and alpha 2C. The Kd value of [3H]-MK 912 for the alpha 2A-subtype was 1.77 nM and for the alpha 2C-subtype 0.075 nM; the receptor sites showing capacities 296 and 33 fmol mg-1 protein, respectively. The Kds of guanfacine were 19.9 and 344 nM, respectively. 2. Binding constants of 26 compounds for the two rat cerebral cortex alpha 2-adrenoceptor subtypes were determined by simultaneous computer modelling of control and guanfacine-masked drug competition curves as well as plain guanfacine competition curves using [3H]-MK912 as labelled ligand (i.e. a '3-curve assay'). Of the tested drugs WB4101, corynanthine, rauwolscine, yohimbine, ARC 239 and prazosin were found to be clearly alpha 2C-selective with selectivities ranging from 16 to 30 fold whereas guanfacine, oxymetazoline, BRL 44408 and BRL 41992 were found to be alpha 2A-selective with selectivities ranging from 9 to 22 fold. 3. The Kds of compounds obtained for the cerebral cortex alpha 2C-adrenoceptors showed an almost 1:1 correlation with the corresponding Kds for alpha 2-adrenoceptors expressed by the pA2d-gene (the rat 'alpha 2-C4' adrenoceptor) in CHO-cells. The cerebral cortex alpha 2A-adrenoceptors did not correlate well with the pA2d alpha 2-adrenoceptor Kds. 4. In the rat spinal cord [3H]-MK 912 bound to alpha 2A- and alpha 2C-adrenoceptor sites with similar affinities as in the cerebral cortex and with densities 172 and 7.4 fmol mg-1 protein, respectively. Drug affinities for some compounds showing major selectivity for alpha 2A- and alpha 2C-adrenoceptors were fully compatible with the notion that the spinal cord sites were alpha 2A- and alpha 2C-adrenoceptors.

Conditions for biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation.

The conditions affording biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation was analyzed theoretically. It was shown that when a competing ligand was subjected to transformation to a ligand that showed higher affinity than the parent compound, biphasic competition curves, which might wrongly be interpreted as indicating the presence of two receptor sites, could be observed in binding assays containing a homogenous receptor population. Biphasic competition curves were seen if the conversion of the competitor occurred according to zero and second order kinetics, as well as by enzymatic catalytic processes. However, when the conversion occurred according to a first order kinetics, the competition curves were uniphasic and resolved only into one-site fits, with the apparent affinity of the competitor reflecting the degree of conversion of the competitor to its metabolite. When the metabolic conversion resulted in a metabolite that showed lower affinity for the receptor than the parent compound, the competition curves became supersteep for conversions according to zero and second order kinetics, as well as for conversion by enzymatic catalytic processes.

Characterization of the enzymatic activity for biphasic competition by guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) at alpha2-adrenoceptors. II. Description of a xanthine-dependent enzymatic activity in spleen cytosol.

The mechanism for formation of high affinity binding of guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) to alpha2-adrenoceptors by the rat spleen cytosol was studied. We report here that the spleen cytosolic fraction mediated the reduction of guanoxabenz to guanabenz (1-(2,6-dichlorobenzylidene-amino)-3-guanidine), the latter having an almost 100-fold higher affinity for rat alpha2A-adrenoceptors than guanoxabenz itself. The reaction product could be separated by high-performance liquid chromatography and its identity as guanabenz confirmed by nuclear magnetic resonance. The spleen cytosolic activity could be separated into high and low molecular weight components, the high molecular weight component requiring low molecular weight factors for maximal activity. Xanthine oxidase seems to be the most likely candidate responsible for the activity, as the guanoxabenz-reducing activity of the high molecular weight component could be sustained by exogenously applied xanthine, while it was potently blocked by allopurinol. The conversion of guanoxabenz by the cytosolic activity was also quite potently blocked by DWO1, 1-(3,4-dimethoxybenzylideneamino)3-hydroxyguanidine, a hydroxyguanidine analogue to guanoxabenz.

Characterization of the enzymatic activity for biphasic competition by guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) at alpha2-adrenoceptors. I. Description of an enzymatic activity in spleen membranes.

The mechanism for formation of high-affinity binding of 1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine (guanoxabenz) to alpha2-adrenoceptors was studied in particulate fractions from the rat spleen. The proportion of apparent high versus low-affinity alpha2-adrenoceptor binding sites increased with increasing incubation time and was also augmented by Mg2+ ions. The formation of high-affinity guanoxabenz binding seemed to be inhibited by a series of N-hydroxyguanidine analogs to guanoxabenz, as well as by a series of metabolic inhibitors that included allopurinol, 1-chloro-2,4-dinitrobenzene, 5,5'-dithiobis-(2-nitrobenzoic acid), cibacron blue, phenyl-p-benzoquinone, didox, and trimidox. The formation of guanoxabenz high-affinity binding was also inhibited in a time- and concentration-dependent fashion by preincubating the membranes with the LW03 N-hydroxyguanidine analogue of guanoxabenz. Moreover, when the spleen membranes were extensively washed for 30 min with buffers at 25 degrees, the guanoxabenz high-affinity binding disappeared. However, when these washed membranes were supplemented with xanthine, the apparent affinity of guanoxabenz increased four to five-fold. Taken together, all data were compatible with the theory that the formation of high-affinity binding was dependent on the generation of a guanoxabenz metabolite that showed an approximate 100-fold greater affinity for the alpha2-adrenoceptors than guanoxabenz itself. Because the most potent blocker of the formation of high-affinity binding was allopurinol (apart from some N-hydroxyguanidine analogs to guanoxabenz) and since the activity could be restored with xanthine, a likely candidate responsible for the metabolic activation is xanthine oxidase.

Autoradiographic studies of central alpha 2A- and alpha 2C-adrenoceptors in the rat using [3H]MK912 and subtype-selective drugs.

In the present study we examined the distribution of alpha 2A- and alpha 2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between alpha 2A- and alpha 2C-adrenoceptors, the slices were incubated with [3H]MK912 in the presence of graded concentrations of the alpha 2A-selective drug, BRL44408, or the alpha 2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM [3H]MK912 plus 185 nM BRL44408 selectively labeled alpha 2C-adrenoceptors, while 0.4 nM [3H]MK912 plus 220 nM spiroxatrine selectively labeled alpha 2A-adrenoceptors. Using this approach, alpha 2C-adrenoceptors were detected in the striatum, while alpha 2A-adrenoceptors predominated in the cortical layers 1-4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus.

Delineation of rat kidney alpha 2A- and alpha 2B-adrenoceptors with [3H]RX821002 radioligand binding: computer modelling reveals that guanfacine is an alpha 2A-selective compound.

We developed a method for the simultaneous determination of drug affinity constants for rat alpha 2A- and alpha 2B-adrenoceptor subtypes by using [3H]RX821002 radioligand binding in the kidney. Three competition curves were obtained for each drug: one for the test compound in the absence of ARC 239 (a drug found to have 108-fold higher affinity for alpha 2B- than for alpha 2A-adrenoceptors), one in the presence of ARC 239, and one for ARC 239. It is possible to determine the Kds of a tested drug for both alpha 2A- and alpha 2B-adrenoceptors by simultaneous computer modelling because of the increased constraint in the calculations given by the inclusion of ARC 239 into the assay. Using this approach, we found guanfacine and oxymetazoline to be highly alpha 2A-selective. The most alpha 2B-selective were ARC 239, prazosin and corynanthine. A number of other drugs, for example UK-14,304, rilmenidine and clonidine, were non-selective or showed minor selectivity for alpha 2A- or alpha 2B-adrenoceptors. Moreover, using Monte Carlo simulations, we showed that the three-curve method gives more accurate estimates of drug binding constants for assays when two receptor sites are present than methods analysing only one competition curve.

Identification of drugs subtype-selective for alpha 2A-, alpha 2B-, and alpha 2C-adrenoceptors in the pig cerebellum and kidney cortex.

The radioligands [3H]MK912 and [3H]RX821002 were used to label alpha2A-, alpha2B-, and alpha2C-adrenoceptors of the pig cerebellum and kidney cortex. By inclusion of the alpha2A-adrenoceptor-selective drug, BRL44408, and using a 'multi-curve' experimental design all the three porcine alpha2-adrenoceptor subtypes could be characterized pharmacologically. The data indicate that the pig alpha2-adrenoceptor subtypes are pharmacologically more related to human alpha2-adrenoceptor subtypes than to the rodent alpha2-adrenoceptors. We suggest a set of drugs that are useful for the delineation of the pig alpha2-adrenoceptor subtypes.

Medetomidine stereoisomers delineate two closely related subtypes of idazoxan (imidazoline) I-receptors in the guinea pig.

A number of imidazoline, imidazole and guanidinium compounds and other drugs were compared for their ability to bind to non-adrenergic idazoxan (imidazoline) I-receptors in particulate guinea pig cerebral cortex and ileum smooth muscle fractions. Radioligand binding with [3H]idazoxan was used for the experiments. Computer modelling of the binding data gave dissociation constants for drug binding to both I-receptors and alpha 2-adrenoceptors. Most drugs showed similar affinities for I-receptors in cortex and ileum. However, medetomidine stereoisomers as well as a few other drugs clearly delineated the I-receptors in cortex and ileum as different.

Characterization of alpha1-adrenoceptor subtypes in the pig.

The identities of the alpha1-adrenoceptor subtypes present in various tissues of the pig were studied using [3H]prazosin radioligand binding. The subtypes were characterized by performing competition experiments for various subtype selective drugs. In the cerebral cortex, spleen and heart, both alpha1A- and alpha1B-adrenoceptors were detected. In the liver was found only the alpha1A-subtype, while in the aorta was found only the alpha1B-subtype. An alpha1-adrenoceptor subtype was present in the adrenal gland with a high affinity for prazosin, the pKd value being 9.6, but with relatively low affinities for other alpha1-adrenoceptor binding drugs. The adrenal gland alpha1-adrenoceptor did not seem to represent the classical alpha1D-subtype, since drugs selective for the alpha1D-subtype in other species, including BMY7378 and SKF104856, showed low affinities for the pig adrenal gland alpha1-adrenoceptor.

[3H]RS79948-197 binding to human, rat, guinea pig and pig alpha2A-, alpha2B- and alpha2C-adrenoceptors. Comparison with MK912, RX821002, rauwolscine and yohimbine.

The Kd values of the recently introduced radioligand [3H]RS79948-197 ((8a R,12aS,13a-S)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-metho xy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine) were determined for the recombinant human and rat alpha2A-, alpha2B- and alpha2C- as well as guinea pig alpha2B- and alpha2c-adrenoceptors expressed in COS (CV-1 Origin, SV40) cells. In addition, the Kd values were also determined for [3H]RS79948-197 for the guinea pig spleen alpha2A-adrenoceptor and for pig alpha2A-, alpha2B- and alpha2C-adrenoceptors in membranes obtained from kidney and striatum. Available radioligands for alpha2-adrenoceptors, besides [3H]RS79948-197 are the tritiated forms of MK912 ((2S,12bS)1',3'-dimethylspiro(1,3,4,5',6,6',7,12b-octa hydro-2H-benzo[b]furo[2,3-a]quinazoline)-2,4'-pyrimidin-2'-one), RX821002 (2-methoxy-idazoxan), rauwolscine and yohimbine. In the present article the binding constants of all these substances for the alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes in human, pig, rat and guinea pig are reviewed. In all species tested MK912 was alpha2C-selective, RX821002 showed a minor alpha2A-selectivity, whereas [3H]RS79948-197 was non-selective among the alpha2-adrenoceptor subtypes, showing high affinity for all three subtypes. Rauwolscine and yohimbine showed relatively low affinities for nmost of the alpha2-adrenoceptor subtypes investigated, the exception being rauwolscine having high affinity for the human and porcine alpha2C-adrenoceptors.

Evidence for the existence of two forms of alpha 2A-adrenoceptors in the rat.

The alpha 2A-adrenoceptors in rat spleen, kidney, spinal cord and cerebral cortex were studied using [3H]-RX821002 radioligand binding. In the spleen, spinal cord and cerebral cortex, the ligand bound to saturable sites with a Kd of about 1 nmol/l and capacities of 134, 240 and 290 fmol/mg protein, respectively. Computer modelling competition curves for 39 drugs, including those for alpha 2A-, alpha 2B- or alpha 2C-adrenoceptor selective drugs, indicated that the sites labelled by [3H]-RX821002 in the spleen consisted of a single population of alpha 2A-adrenoceptors. However, the competition curves for guanoxabenz were definitely biphasic and resolved into two site fits, indicating that guanoxabenz was binding to both high affinity (Kd = 35 nmol/l) and low affinity (Kd = 8900 nmol/l) alpha 2A-adrenoceptor sites in the proportions 57% and 43%, respectively. The KdS for a number of alpha 2-adrenoceptor subtype selective drugs, measured in competition with [3H]-RX821002 in cerebral cortex and spinal cord, were highly correlated with those obtained in the spleen indicating their alpha 2A-adrenoceptor nature. However, by contrast to the results with the spleen, the guanoxabenz competition curves for the spinal cord and cerebral cortex were monophasic and resolved only into one site fits, the Kd of guanoxabenz being about 4000 nmol/l for both tissues. Drug KdS for kidney alpha 2A-adrenoceptors were also determined using [3H]-RX821002. For nearly all drugs tested, the KdS were highly correlated with those found for the alpha 2A-adrenoceptors in the other rat tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

[Treatment of acute diarrhea: prescription patterns by private practice pediatricians]. / Traitement des diarrhées aiguës: les habitudes de prescription des pédiatres libéraux.

UNLABELLED: Acute gastroenteritis remains a frequent illness in infants and children with still important morbidity and mortality rates. Oral rehydratation solutions (ORS) and early refeeding are the main recommendations. Indication of drugs remains limited. OBJECTIVE: To evaluate the management of acute diarrhea by private practice pediatricians of France. METHODS: A questionnaire concerning ORS, dietary formula, antidiarrheal diet, antibiotherapy, antidiarrheal drugs was sent to all 2907 private pediatricians of France. RESULTS: Six hundred twenty-nine questionnaires were analyzed (22%). Three hundred and ninety-seven pediatricians (63%) prescribed systematically an ORS, 294 (47%) changed formula, 412 (66%) prescribed a regimen. Antibiotic was prescribed after coproculture (81%), when glairy and bloody diarrhea (65%), associated infectious disease (63%), toxi-infectious syndrome (42%) or immunodeficiency were present (28%). Most pediatricians (97%) prescribed at least one drug: diosmectite (84%), Lactobacillus acidophilus (63%), Saccharomyces boulardii (62%), racecadotril (62%), loperamide (28%), attapulgite de Mormoiron (26%), nifuroxazide (20%). Drugs were prescribed more often for their effectiveness than for comfort. CONCLUSION: This study demonstrates the discrepancies that remain between recommendations and practical care in the treatment of acute diarrhea in children. Private French pediatricians often prescribe drugs.

Mitomycin C: an alternative conservative treatment for refractory esophageal stricture in children?

BACKGROUND AND STUDY AIMS: Mitomycin C is an antiproliferative agent that has been used successfully as an adjunct treatment in ophthalmological procedures, in the management of laryngeal and tracheal stenosis, and more recently to prevent the recurrence of caustic esophageal strictures in children. The aim of this study was to assess the efficacy and safety of local application of mitomycin C to refractory esophageal strictures in children. PATIENTS AND METHODS: We performed a preliminary prospective study of local application of mitomycin C in four children, aged between 1 year and 6 years, who had refractory esophageal strictures. Two of the children presented with strictures caused by caustic ingestion and the other two children had anastomotic strictures following surgical repair of congenital esophageal atresia. The patients had required between four and ten esophageal dilations over a 5-24-month period before mitomycin C application. After an endoscopic dilation, mitomycin C was applied onto the dilation wound using a rigid endoscope. RESULTS: No complications were observed after the procedure. One child required a second application of mitomycin C 2 weeks after the first application because of recurrence of dysphagia. All the children remained asymptomatic and none of them required further dilation over a mean follow-up period of 24 months. Radiological control examinations revealed that there was no recurrence of the esophageal strictures and esophageal biopsies performed during follow-up showed no signs of dysplasia. CONCLUSIONS: Local application of mitomycin C is a potential alternative to iterative dilations, surgery, or stent placement for the treatment of refractory esophageal strictures in children. However, prospective, long-term assessment of outcomes is needed before any definitive conclusion can be drawn about the usefulness of mitomycin C in these patients.

Inhibition of cyclic AMP production by alpha 2-adrenoceptor stimulation in the guinea-pig spinal cord slices.

In spinal cord slices isolated from guinea-pig and preincubated with 3H-adenine, 0.3-30 microM forskolin induced a dose-dependent increase in the content of 3H-cAMP, the maximal increase being about 8-fold. The selective alpha 2-adrenergic agonist UK-14,304 (10 microM) reduced both the basal and the forskolin stimulated levels of 3H-cAMP by 18-32%. Dose response curves of the effect of UK-14,304 on cAMP production in the spinal cord slices, stimulated with 3 microM forskolin, showed an IC50 of 37 nM and a maximally inhibitory effect of 27%. A number of other alpha 2-adrenergic agonist (clonidine, guanfacine, B-HT 920 and B-HT 933) also inhibited the forskolin stimulated 3H-cAMP production; clonidine and guanfacine being almost equipotent with UK-14,304, but their maximal inhibitory effects being only about 6-7%. B-HT 920 and B-HT 933 were less potent and their maximal inhibitory effects about 16-21%. The dose response curve of UK-14,304 on inhibition of forskolin stimulated cAMP production was shifted almost 50-fold to the right by 0.3 microM yohimbine. Prazosin (0.3 microM) did not affect the UK-14,304 dose response curve. It is concluded that alpha 2-adrenoceptor stimulation mediates inhibition of cAMP production in the guinea-pig spinal cord.

Relationship between forskolin and calcium-calmodulin stimulation of rat cerebral cortex adenylate cyclase: enzyme activation modulates substrate (MgATP) affinity.

In membranes prepared from the rat cerebral cortex EGTA 0.4-100 mumol/l was found to dose-dependently inhibit adenylate cyclase activity, both during basal conditions and when the cyclase activity had been stimulated with 10 mumol/l forskolin. Addition of calcium 2-30 mumol/l (in excess of EGTA) totally prevented the inhibition induced by EGTA, both in the absence and presence of forskolin. These data are consistent with the notion that in the absence of EGTA the rat cortex adenylate cyclase is considerably stimulated by endogenous calcium-calmodulin. The data also show that stimulation with endogenous calcium-calmodulin is more than additive with that of forskolin indicating an action on identical cyclase units. The kinetics of the rat cortex adenylate cyclase for its substrate (MgATP) was also investigated. Activation of the cyclase with endogenous calcium-calmodulin, induced a marked increase in the Vmax of the enzyme and a concomitant almost 2-fold increase in the Km. Stimulation with 10 microM forskolin also induced a marked increase in the Vmax as well as an almost 3-fold increase in the Km. A combination of the two stimulants caused a further increase in Vmax as well as Km; the Vmax being increased more than 17-fold and the Km being increased 3.5-fold over the basal. The data indicates that both calcium-calmodulin and forskolin induces a similar type of activation of the adenylate cyclase which is associated with a lowered affinity for its substrate.