Biomedical subjects

F M Rogerson

Publications and source records attributed to F M Rogerson.

At least 19 recordsLinked to original sources

PubMed2003

Determinants of spironolactone binding specificity in the mineralocorticoid receptor.

Spironolactone is a mineralocorticoid receptor (MR) antagonist in clinical use. The compound has a very low affinity for the glucocorticoid receptor (GR). Determinants of binding specificity of spironolactone to the MR were investigated using chimeras created between the ligand-binding domains (LBDs) of the MR and the GR. These chimeras had previously been used to investigate aldosterone binding specificity to the MR. Spironolactone was able to compete strongly for [(3)H]-aldosterone and [(3)H]-dexamethasone binding to a chimera containing amino acids 804-874 of the MR, and weakly for [(3)H]-dexamethasone binding to a chimera containing amino acids 672-803 of the MR. Amino acids 804-874 were also critical for aldosterone binding specificity. Models of the MR LBD bound to aldosterone and spironolactone were created based on the crystal structure of the progesterone receptor LBD. The ligand-binding pocket of the MR LBD model consisted of 23 amino acids and was predominantly hydrophobic in nature. Analysis of this model in light of the experimental data suggested that spironolactone binding specificity is not governed by amino acids in the ligand-binding pocket.

Aldosterone↗

PubMed2000

Mineralocorticoid action.

The physiology of mineralocorticoid action, particularly with respect to epithelial sodium transport, is well defined. A full understanding of the molecular basis of mineralocorticoid action has however proven to be more elusive. In the last decade insights into structural and functional aspects of the mineralocorticoid receptor combined with emerging details of the components of the mediators of the sodium flux has resulted in a clearer picture. This review focuses on two aspects of these new developments; the mineralocorticoid receptor and putative aldosterone induced proteins.

Aldosterone↗

PubMed1999

Structural determinants of aldosterone binding selectivity in the mineralocorticoid receptor.

The structural determinants of aldosterone binding specificity in the mineralocorticoid receptor (MR) have not been determined. The MR has greatest sequence identity with the better characterized glucocorticoid receptor (GR), which is reflected in their overlapping ligand binding specificities. There must be subtle sequence differences that can account for the MR-specific binding of aldosterone and the shared binding of cortisol. To characterize ligand binding specificity, chimeras were made between the human MR and GR ligand-binding domains (LBDs). Three points were chosen as break points to generate a total of 16 different constructs. These chimeric LBDs were placed in a human GR expression vector containing the GR DNA-binding and N-terminal domains and assayed by co-transfection into CV-1 cells with the mouse mammary tumor virus-luciferase reporter plasmid. Binding of [(3)H]aldosterone and [(3)H]dexamethasone was also measured. All of the constructs that are potently activated by aldosterone contain amino acids 804-874 of the MR. The results of the ligand binding experiments using [(3)H]aldosterone were consistent with the transactivation assay. Cortisol activation of the chimeras was surprisingly complex. Constructs that are activated by cortisol contain either amino acids 804-874 and 932-984 of the MR or amino acids 598-668 and 726-777 of the GR. However, all of the chimeras retained the ability to bind the synthetic glucocorticoid [(3)H]dexamethasone, and cortisol was able to displace [(3)H]dexamethasone binding, suggesting that the differential effects of cortisol on transcriptional activation are caused by an effect that occurs downstream of ligand binding. These results identify a subregion of the MR LBD that confers specificity of aldosterone binding, which contrasts with cortisol binding where differential effects between chimeras appear to be mediated by interactions distal to ligand binding.

Aldosterone↗

PubMed1999

Mutants of 11beta-hydroxysteroid dehydrogenase (11-HSD2) with partial activity: improved correlations between genotype and biochemical phenotype in apparent mineralocorticoid excess.

Mutations in the kidney isozyme of human 11-hydroxysteroid dehydrogenase (11-HSD2) cause apparent mineralocorticoid excess, an autosomal recessive form of familial hypertension. We studied 4 patients with AME, identifying 4 novel and 3 previously reported mutations in the HSD11B2 (HSD11K) gene. Point mutations causing amino acid substitutions were introduced into a pCMV5/11HSD2 expression construct and expressed in mammalian CHOP cells. Mutations L179R and R208H abolished activity in whole cells. Mutants S180F, A237V, and A328V had 19%, 72%, and 25%, respectively, of the activity of the wild-type enzyme in whole cells when cortisol was used as the substrate and 80%, 140%, and 55%, respectively, of wild-type activity when corticosterone was used as the substrate. However, these mutant proteins were only 0.6% to 5.7% as active as the wild-type enzyme in cell lysates, suggesting that these mutations alter stability of the enzyme. In regression analyses of all AME patients with published genotypes, several biochemical and clinical parameters were highly correlated with mutant enzymatic activity, demonstrated in whole cells, when cortisol was used as the substrate. These included the ratio of urinary cortisone to cortisol metabolites (R(2)=0.648, P<0.0001), age at presentation (R(2)=0.614, P<0.0001), and birth weight (R(2)=0.576, P=0.0004). Approximately 5% conversion of cortisol to cortisone is predicted in subjects with mutations that completely inactivate HSD11B2, suggesting that a low level of enzymatic activity is mediated by another enzyme, possibly 11-HSD1.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1998

Characterization of cDNAs encoding isoforms of hamster 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase.

Western blot analyses of various hamster tissues reveal high levels of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in adrenal and liver, and moderate levels of expression in kidney. The expression in liver is sexually dimorphic; very high levels of protein are observed in adult male liver but very low levels are seen in the female liver. Three distinct cDNAs encoding isoforms of 3 beta-HSD were isolated from hamster cDNA libraries. The type 1 isoform is a high-affinity dehydrogenase/isomerase expressed in adrenal and male kidney. The type 2 isoform is also a high-affinity dehydrogenase/isomerase expressed in kidney and male liver. The type 3 enzyme is a 3-ketosteroid reductase expressed predominantly in kidney. Sequencing of the clones showed that all three are structurally very similar, although types 1 and 2 share the greatest degree of similarity. Immunohistochemical staining for 3 beta-HSD in the adrenal was found throughout the adrenal cortex. In the kidney staining was confined to tubules, and in the liver, heavy staining was found in hepatocytes. The cloning of cDNAs for 3 beta-HSD from the liver and kidney should help in elucidating the function of this enzyme in these tissues.

Adrenal Glands↗

PubMed1997

Variation in placental type 2 11beta-hydroxysteroid dehydrogenase activity is not related to birth weight or placental weight.

It has been suggested that the association between the development of hypertension and a combination of low birth weight and high placental weight can be explained by variations in expression of NAD+-dependent 11beta-hydroxysteroid dehydrogenase (11-HSD2 or 11-HSD K) in the placenta. Enzymatic activity and mRNA levels of 11-HSD2 were measured in 111 human placentas taken from normal births. There were no correlations between either 11-HSD2 activity or mRNA levels and either fetal or placental weight. These studies suggest that variations in placental 11-HSD activity do not influence fetal or placental weight in humans.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1997

Molecular analysis of 11 beta-hydroxysteroid dehydrogenase and its role in the syndrome of apparent mineralocorticoid excess.

The syndrome of apparent mineralocorticoid excess (AME) is an inherited form of hypertension in which 11 beta-hydroxysteroid dehydrogenase (11-HSD) is defective. This enzyme converts cortisol to its inactive metabolite, cortisone. The deficiency allows mineralocorticoid receptors to be occupied by cortisol, because these receptors themselves have similar affinities for cortisol and aldosterone. There are two isozymes of 11-HSD, a liver (L) or type 1 isozyme with a relatively low affinity for steroids, and a kidney (K) or type 2 isozyme with high steroid affinity. Mutations in the gene for the kidney isozyme of 11-HSD have been detected in all kindreds with AME. We expressed enzymes carrying all known missense mutations in cultured cells and determined their activity. For each patient with AME, we compared the enzymatic activity predicted by the genotype with the ratio of cortisol to cortisone metabolites in the urine, (THF + aTHF)/THE. These were strongly correlated, suggesting that the biochemical phenotype of AME is largely determined by genotype. The K isozyme of 11-HSD is also expressed in high levels in the placenta, where its function is unclear. AME patients often have low birth weight. By analogy with AME, low placental 11-HSD K activity in humans might be a risk factor for low birth weight and subsequent hypertension. However, we found that there was no significant correlation between 11-HSD activity, mRNA levels, and either fetal or placental weight.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1997

11 beta-Hydroxysteroid dehydrogenase and its role in the syndrome of apparent mineralocorticoid excess.

Aldosterone, the most important mineralocorticoid, regulates electrolyte excretion and intravascular volume mainly through its effects on renal distal tubules and cortical collecting ducts, where it acts to increase sodium resorption from and potassium excretion into the urine. Excess secretion of aldosterone or other mineralocorticoids, or abnormal sensitivity to mineralocorticoids, may results in hypokalemia, suppressed plasma renin activity, and hypertension. The syndrome of apparent mineralocorticoid excess (AME) is an inherited form of hypertension in which 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) is defective. This enzyme converts cortisol to its inactive metabolite, cortisone. Because mineralocorticoid receptors themselves have similar affinities for cortisol and aldosterone, it is hypothesized that the deficiency allows these receptors to be occupied by cortisol, which normally circulates at levels far higher than those of aldosterone. We cloned cDNA and genes encoding two isozymes of 11 beta-HSD. The liver (L) or type 1 isozyme has relatively low affinity for steroids, is expressed at high levels in the liver but poorly in the kidney, and is not defective in AME. The kidney (K) or type 2 isozyme has high steroid affinity and is expressed at high levels in the kidney and placenta. Mutations in the gene for the latter isozyme have been detected in all kindreds with AME. Moreover, the in vitro enzymatic activity conferred by each mutation is strongly correlated with the ratio of cortisol to cortisone metabolites in the urine [tetrahydrocortisone (THF) +allo-THF]/THE. This suggests that the biochemical phenotype of AME is largely determined by genotype.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1997

Alternate splicing of the RNA for hamster type 2 3 beta-hydroxysteroid dehydrogenase/delta 5-->4isomerase.

Complementary DNAs encoding the hamster type 2 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase were isolated from liver and kidney cDNA libraries. Nine clones were isolated containing identical coding and 3' untranslated sequences. However, six of the clones contained a 68-nucleotide stretch in the 5' untranslated region that was missing in the other three clones. Primers were designed to flank this region and the polymerase chain reaction (PCR) was performed on hamster liver and adrenal RNA. Two PCR products were amplified of the predicted molecular sizes and with the expected sequence. Primers were then designed to amplify sequences encompassing this region from hamster genomic DNA. Sequencing of the resultant PCR products demonstrated that the 68-nucleotide stretch missing in some transcripts corresponded exactly to the second of three exons identified. We conclude that the 5' untranslated region of this mRNA is transcribed from at least three exons, and that the sequence of the second of these exons is spliced out of some of the RNA transcripts.

Alternative Splicing↗

PubMed1995

Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11 beta-hydroxysteroid dehydrogenase.

11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) converts glucocorticoids to inactive products and is thus thought to confer specificity for aldosterone on the type I mineralocorticoid receptor in the kidney. Recent studies indicate the presence of at least two isozymes of 11 beta HSD. In vitro, the NAD(+)-dependent kidney (type 2) isozyme catalyzes 11 beta-dehydrogenase but not reductase reactions, whereas the NADP(+)-dependent liver (type 1) isozyme catalyzes both reactions. We have now characterized the human gene encoding kidney 11 beta HSD (HSD11K). A bacteriophage P1 clone was isolated after screening a human genomic library by hybridization with sheep HSD11K cDNA. The gene consists of 5 exons spread over 6 kb. The nucleotide binding domain lies in the first and the second exon, and the catalytic domain in the fourth exon. The 5' flanking sequences and first exon are GC-rich (80%), suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescence in situ hybridization of metaphase chromosomes with a positive P1 clone localized the gene to chromosome 16q22. In contrast, the HSD11L (liver isozyme) gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. HSD11K is expressed at high levels in the placenta and kidney of midgestation human fetuses and at lower levels in lung and testes. Different transcriptional start sites are utilized in kidney and placenta. These data should be applicable to genetic analysis of the syndrome of apparent mineralocorticoid excess, which may represent a deficiency of 11 beta HSD.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1995

Localization of angiotensin converting enzyme by in vitro autoradiography in the rabbit brain.

The distribution of angiotensin converting enzyme was examined in the rabbit brain by in vitro autoradiography with the specific radiolabelled inhibitor 125I-351A. In the rabbit, the highest concentrations of radioligand binding were found in the choroid plexus, blood vessels, subfornical organ, vascular organ of the lamina terminalis, area postrema and inferior olive. High levels of binding were found throughout the basal ganglia, consistent with the results in all other species studied. In the midbrain the central gray and the superior colliculus displayed high levels of binding. In the medulla oblongata high levels of binding were associated with the nucleus of the solitary tract and dorsal motor nucleus of vagus, consistent with the pattern in other species. There was moderate labelling throughout both the cerebral and cerebellar cortices, which contrasts to the rat but is consistent with the situation in primates. Angiotensin converting enzyme (ACE) is more widely distributed in rabbit brain that in rat, human and Macaca fascicularis, and the results suggest ACE has a very general role in the metabolism of neuropeptides. Inhibitors of converting enzyme are very widely used in the treatment of hypertension and heart disease, and the rabbit should provide a useful model for examining the effects of these drugs in the brain.

Animals↗

PubMed1995

Analysis of the human gene encoding the kidney isozyme of 11 beta-hydroxysteroid dehydrogenase.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyzes the conversion of cortisol to cortisone. This activity may be deficient in the syndrome of apparent mineralocorticoid excess (AME). 11 beta-HSD L (Type I), isolated from liver, is widely expressed and utilizes NADP+ as a cofactor. The gene for 11 beta-HSD L was found to be normal in patients of AME. A second isoform, 11 beta-HSD K (Type II), isolated from kidney, is more tissue specific in expression and utilizes NAD+ as a cofactor. The cDNA clone encoding 11 beta-HSD K was isolated from sheep kidney. The cDNA is 1.8 kb in length and encodes a protein of 404 amino acid residues with a predicted M(r) 43,953. The recombinant enzyme functions as an NAD(+)-dependent 11 beta-dehydrogenase with very high affinity for steroids, but it has no detectable reductase activity. It is 37% identical in amino acid sequence to an NAD(+)-dependent isozyme of 17 beta-hydroxysteroid dehydrogenase. It is expressed at high levels in the kidney, placenta, adrenal and at lower levels in colon, stomach, heart and skin. The human 11 beta-HSD K gene consists of five exons spread over 6 kb. The nucleotide binding domain lies in the first and the second exon, and the catalytic domain in the fourth exon. The promoter for 11 beta-HSD K gene lacks a TATA box and has a high GC base content, suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescent in situ hybridization of metaphase chromosomes with a positive bacteriophage P1 genomic 11 beta-HSD K clone localized the gene to chromosome 16q22. In contrast, the 11 beta-HSD L gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. Different transcriptional start sites are utilized in kidney and placenta.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1995

Expression and characterization of isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in the hamster.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta-HSD) is essential for the production of all classes of steroid hormones. Multiple isozymes of this enzyme have been demonstrated in the kidney and liver of both the rat and the mouse, although the function of the enzyme in these tissues is unknown. We have characterized three isozymes of 3 beta-HSD expressed in various tissues of the hamster. Both western and northern blot analyses demonstrated very high levels of 3 beta-HSD in the adrenal, kidney and male liver. Conversely, there were extremely low levels of enzyme expression in the female liver. cDNA libraries prepared from RNA isolated from hamster adrenal, kidney and liver were screened with a full-length cDNA encoding human type 1 3 beta-HSD. Separate cDNAs encoding three isoforms of 3 beta-HSD were isolated from these libraries. To examine the properties of the isoforms, the cDNAs were ligated into expression vectors for over-expression in 293 human fetal kidney cells. The type 1 isoform, isolated from an adrenal cDNA library, was identified as a high-affinity 3 beta-hydroxysteroid dehydrogenase. A separate isoform, designated type 2, was isolated from the kidney, and this was also a high-affinity dehydrogenase/isomerase. Two cDNAs were isolated from the liver, one identical in sequence to type 2 of the kidney, and a distinct cDNA encoding an isoform designated type 3. The type 3 3 beta-HSD possessed no steroid dehydrogenase activity but was found to function as a 3-ketosteroid reductase. Thus male hamster liver expresses a high-affinity 3 beta-HSD (type 2) and a 3-ketosteroid reductase (type 3), whereas the kidney of both sexes express the type 2 3 beta-HSD isoform. These differ from the type 1 3 beta-HSD expressed in the adrenal cortex.

Amino Acid Sequence↗

PubMed1995

Human hypertension caused by mutations in the kidney isozyme of 11 beta-hydroxysteroid dehydrogenase.

The syndrome of apparent mineralocorticoid excess (AME) is an inherited form of human hypertension thought to result from a deficiency of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). This enzyme normally converts cortisol to inactive cortisone and is postulated to thus confer specificity for aldosterone upon the mineralocorticoid receptor. We have analysed the gene encoding the kidney isozyme of 11 beta HSD and found mutations on both alleles in nine of 11 AME patients (eight of nine kindreds). These mutations markedly affect enzymatic activity. They thus permit cortisol to occupy the renal mineralocorticoid receptor and thereby cause sodium retention and hypertension.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1995

Type 2 11 beta-hydroxysteroid dehydrogenase messenger ribonucleic acid and activity in human placenta and fetal membranes: its relationship to birth weight and putative role in fetal adrenal steroidogenesis.

Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) have been described which catalyze the interconversion of cortisol (F) to cortisone (E). 11 beta HSD activity has previously been reported in placenta and fetal membranes, where its role may be to protect the developing fetus from glucocorticoid excess. Furthermore, in the rat, an association between placental 11 beta HSD activity and the subsequent development of hypertension in the offspring has been reported. We have characterized the isoforms of 11 beta HSD in human fetal membranes and dissected placental tissue at term and investigated the relationship between placental 11 beta HSD activity and fetal and placental weights. 11 beta HSD activity studies in the presence of 0.1 mumol/L F and NAD (indicative of type 2 isoform activity) revealed high levels of activity in trophoblast dissected free of vessels (561 +/- 87 pmol E/h.mg protein; n = 4) > undissected placenta > cotyledenous vessels dissected away from trophoblast > placental and reflected amnion. In contrast, in the presence of 2.5 mumol/L F and NADP (indicative of type 1 isoform activity), only decidua and chorion demonstrated significant levels of 11 beta HSD activity. Type 1 11 beta HSD activity in chorion was probably due to decidual contamination, in that it was absent in decidua-free fused chorion obtained from a twin pregnancy. In keeping with these data, type 1 11 beta HSD messenger ribonucleic acid (1.5 kilobases) was detected in decidua, but in no other tissue, and high levels of type 2 11 beta HSD messenger ribonucleic acid (1.9 kilobases) were found in undissected placenta and trophoblast. In 27 term placentas, 11 beta HSD activity varied from 194-448 pmol E/h.mg protein. There was a weak, but significant, positive correlation between term placental 11 beta HSD activity and fetal weight (r = 0.408; P = 0.034), but no correlation with placental weight. Thus, in man, the reported association of a small fetus and a large placenta predisposing to adult hypertension cannot be explained on the basis of defective 11 beta HSD activity. However, the placenta offers an immense reservoir for F clearance (1.73-7.95 mumol/min.placenta) and may be a principal factor driving fetal ACTH secretion and, hence, fetal adrenal steroidogenesis.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

Evidence of a steroidogenic enzyme gene dose effect on adrenal gene expression in hereditary rabbit congenital adrenal hyperplasia.

We previously reported the gene deletion encoding cytochrome P-450 cholesterol side-chain cleavage enzyme (P-450SCC, resulting in complete elimination of the adrenal gene expression and causing congenital adrenal hyperplasia in the rabbit. Using the rabbit congenital adrenal hyperplasia model, we investigated the wild type (wt) P-450SCC gene dose effect on gene expression in three P-450SCC genotype animals [wt/wt, wt/mutant (mt), mt/mt] identified by Southern blot analysis. Northern blots using a rabbit P-450SCC cDNA probe revealed no detectable P-450SCC mRNA in individual adrenals of animals with congenital adrenal hyperplasia (mt/mt) and approximately half or slightly less than half the levels of the mRNA in the pooled adrenals of five heterozygous (wt/mt) newborn animals compared with the mRNA levels in the pooled adrenals of five homozygous normal (wt/wt) newborn animals. Identical P-450SCC mRNA levels were found individual adrenals of adult animals with regard to the P-450SCC genotype, although at a higher expression level than in the newborn animals of the same genotype. Control Northern blots using human CPY21-B cDNA and cytoplasmic actin cDNA probes confirmed the accuracy and integrity of RNA. Western immunoblotting using anti-ovine P-450SCC antibody revealed decreased P-450SCC protein in the adrenals of wt/mt animals at approximately half the level of the P-450SCC protein in the adrenals of the wt/wt animals. Baseline and ACTH-stimulated serum corticosterone (B) levels in vivo were similar between the age-matched wt/mt and wt/wt animals, whereas ACTH-stimulated B levels in adult animals were higher than those in the newborn animals irrespective of P-450SCC genotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1992

Presence of angiotensin converting enzyme in the adventitia of large blood vessels.

BACKGROUND: Angiotensin converting enzyme (ACE) is present in the endothelial cells of all vascular beds. There are, however, many reports of converting enzyme activity in blood vessels not associated with the endothelium. METHODS: ACE was localized in large blood vessels of a number of mammals by in vitro autoradiography using the radioligand 125I-351A. To characterize this binding further, immunohistochemistry was performed on rabbit aorta using polyclonal antisera raised to two different preparations of rabbit lung ACE. RESULTS: In all of the blood vessels studied, which included the rabbit pulmonary artery, rabbit, dog and sheep aorta, human internal mammary artery and human saphenous vein, high levels of radioligand binding were found in endothelial cells, as expected. In addition, a very high density of punctate binding was observed interspersed between diffuse moderate labelling in the adventitia. Immunoreactivity was confined to the endothelium of both the intima and the vasa vasorum of the adventitia. The immunostaining correlated well with the autoradiography. The ACE inhibitors lisinopril and perindoprilat displayed similar high affinities in competing for the binding of 125I-351A to the endothelium and adventitia of the sheep aorta, suggesting that at these two sites the radioligand was binding to ACE. CONCLUSIONS: We find that ACE in the adventitia of large blood vessels is confined to the vaso vasorum. The results of this study help to explain the findings of many studies that ACE activity persists in endothelium-denuded blood vessels and also reveals a source of ACE distant from the luminal endothelial surface.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1990

A novel substance P binding site in bovine adrenal medulla.

Radioligand binding techniques were used to characterize the substance P (SP) binding site on membranes prepared from bovine adrenal medullae. 125I-labelled Bolton-Hunter substance P (BHSP), which recognises the C-terminally directed, SP-preferring NK1 receptor, showed no specific binding. In contrast, binding of [3H]SP was saturable (at 6 nM) and reversible, with an equilibrium dissociation constant (Kd) 1.46 +/- 0.73 nM, Bmax 0.73 +/- 0.06 pmol/g wet weight and Hill coefficient 0.98 +/- 0.01. Specific binding of [3H]SP was displaced by SP greater than neurokinin A (NKA) greater than SP(3-11) approximately SP(1-9) greater than SP(1-7) approximately SP(1-4) approximately SP(1-6), with neurokinin B (NKB) and SP(1-3) very weak competitors and SP(5-11), SP(7-11) and SP(9-11) causing negligible inhibition (up to 10 microM). This potency order is quite distinct from that seen with binding to an NK1 site, a conclusion confirmed by the lack of BHSP binding. It appears that Lys3 and/or Pro4 are critical for binding, suggesting an anionic binding site. These data suggest the existence of an unusual binding site which may represent a novel SP receptor. This site appears to require the entire sequence of the SP molecule for full recognition.

Adrenal Medulla↗