Interobserver and Intraobserver Reliability in the Radiologic Assessment of Lumbar Interbody Fusion.

STUDY DESIGN: Retrospective cohort study comparing intraobserver and interobserver reliability of 3 different radiologic fusion classifications following uninstrumented single-level anterior lumbar interbody fusion. OBJECTIVE OF THE STUDY: The objective of the study was to compare the intraobserver and interobserver reliability of 3 different radiologic spinal fusion scoring systems. SUMMARY OF BACKGROUND DATA: Knowledge regarding radiologic spinal fusion is crucial when studying patients that were treated with lumbar interbody fusion. The scoring system should be reliable and reproducible. Various radiologic classification systems coexist, but the reliability of these systems has thus far not been compared in a single consecutive group of patients. The aim of the present study was the identification of the most valid scoring system in the assessment of interbody fusion. METHODS: We studied a retrospective consecutive cohort of 50 patients who underwent an anterior lumbar interbody fusion procedure by a single surgeon using a stand-alone cage performed between 1993 and 2002. Plain anterior-posterior, lateral radiographs, and flexion-extension radiographs were made during follow-up visits and were used for analysis. The interbody fusion was scored on these radiographic images using the 3 classification systems (Brantigan, Burkus, and the Radiographic Score) by 2 experienced musculoskeletal radiologists and 2 senior orthopedic spinal surgeons all of whom were blinded to clinical data and outcome. RESULTS: Of the 3 classifications included in the current study, the Burkus classification had a moderate interobserver agreement and a substantial to perfect intraobserver agreement. The other classifications (Bratingan and the Radiographic Score) showed only fair interobserver agreement and moderate to substantial agreement among all observers. No significant differences in reliability between orthopedic surgeons and radiologists were found for all 3 classifications. CONCLUSIONS: The Burkus classification system was classified as most reliable in this, but showed only moderate interobserver agreement. Therefore, the need for a more reliable classification system for the radiographic assessment of lumbar interbody fusion still exists to date.

Spinal decompensation in degenerative lumbar scoliosis.

Due to the aging population, degenerative scoliosis is a growing clinical problem. It is associated with back pain and radicular symptoms. The pathogenesis of degenerative scoliosis lies in degenerative changes of the spinal structures, such as the intervertebral disc, the facet joints and the vertebrae itself. Possibly muscle weakness also plays a role. However, it is not clear what exactly causes the decompensation to occur and what determines the direction of the curve. It is known that in the normal spine a pre-existing rotation exists at the thoracic level, but not at the lumbar level. In this retrospective study we have investigated if a predominant curve pattern can be found in degenerative scoliosis and whether symptoms are predominantly present at one side relative to the curve direction. The lumbar curves of 88 patients with degenerative scoliosis were analyzed and symptoms were recorded. It was found that curve direction depended significantly on the apical level of the curve. The majority of curves with an apex above L2 were convex to the right, whereas curves with an apex below L2 were more frequently convex to the left. This would indicate that also in degenerative scoliosis the innate curvature and rotational pattern of the spine plays a role in the direction of the curve. Unilateral symptoms were not coupled to the curve direction. It is believed that the symptoms are related to local and more specific degenerative changes besides the scoliotic curve itself.

Natriuretic peptide receptors of type A in human neuroblastomas.

Functional natriuretic peptide receptors of type A (NPR-A) were detected in the human neuroblastoma NB-OK-1, SK-N-SH and SK-N-BE, but not the SH-SY5Y, cell lines. Also, NPR-A mRNA was detected in 19 of the 25 tumor neuroblastoma samples tested in this study. Five of the eight tumor neuroblastoma samples that were assayed for atrial natriuretic peptide (ANP) binding revealed the presence of ANP-binding sites. In the human neuroblastoma NB-OK-1 cell line, [(3)H] thymidine incorporation was increased in response to ANP, decreased in response to pituitary adenylate cyclase-activating polypeptide (PACAP-27), and the stimulatory effect of ANP was inhibited by PACAP-27. Tissue transglutaminase activity was decreased by ANP and PACAP-27, and their effects were additive. However, neither cell cycle phases, cell growth, or cell apoptosis were modified by ANP or PACAP-27 treatments.

Human A-type ANP receptor upregulation by PACAP and carbachol in neuroblastoma cells.

Pituitary adenylyl cyclase activating polypeptide (PACAP-27), forskoline and carbachol increased type A atrial natriuretic peptide receptor (NPR-A) density, as well as NPR-A mRNA level, in the human neuroblastoma NB-OK-1 cell line. TPA did not have any effect per se, but blunted the effect of PACAP-27 on both NPR-A density and NPR-A mRNA. The half-life of the NPR-A mRNA was not modified by any of the agents tested. Our data support an original transcriptional upregulation of human NPR-A in response to cAMP-induced agents, and in response to carbachol.

Ferritin-associated iron induces neutrophil dysfunction in hemosiderosis.

Neutrophils (PMNs) from patients with secondary iron overload have an increased iron and ferritin content as well as a phagocytosis defect. Several serum components might be incriminated in the cellular iron accumulation. We therefore compared the effects on the PMN phagocytosis of total serum as well as the ferritin and transferrin fractions of serum derived from patients with thalassemia major and healthy control subjects. An incubation system of PMNs was developed. PMN phagocytosis was measured before and after incubation. Total serum from patients with thalassemia induced a defect that was prevented by co-incubation with deferoxamine (DFO). Gel-filtration chromatography was performed to separate the serum fraction containing transferrin and albumin from that containing ferritin. The transferrin-albumin fraction had no effect on PMN phagocytosis. On the contrary, the ferritin fraction of normal serum was deleterious to PMN phagocytosis, and the same fraction from thalassemic serum decreased PMN phagocytosis even more. Co-incubation with DFO or catalase improved this defect. Moreover, a cellular increase in the L-type subunit of ferritin was observed after the incubation of PMNs with the ferritin-containing fraction from thalassemic serum. In conclusion, serum from patients with thalassemia is toxic to PMNs, and this toxicity is due to ferritin-associated iron.

The pituitary adenylate cyclase activating polypeptide (PACAP I) and VIP (PACAP II VIP1) receptors stimulate inositol phosphate synthesis in transfected CHO cells through interaction with different G proteins.

The PACAP receptor (PACAP I receptor, selective for PACAP) and the PACAP II VIP1 receptor (recognizing PACAP and VIP with the same high affinity) were stably expressed in Chinese Hamster Ovary (CHO) cells. Cell lines expressing different receptor densities, as measured by binding saturation curves, were selected. Inositol phosphate production was stimulated dose dependently in all the cell lines by PACAP and VIP, and the order of potency of the agonists was identical to that of high affinity receptor occupancy. The stimulatory effect of a saturating peptide concentration was proportional to the total receptor density. At similar receptor densities, however, the PACAP receptor mediated stimulation was higher than the VIP receptor-mediated stimulation. Pretreatment of the cells with pertussis toxin for 8 h had no effect on receptor densities, did not alter the PACAP stimulated inositol phosphate synthesis by the cells expressing the PACAP I receptor but markedly inhibited the response of the cells expressing the PACAP II VIP1 receptor. Thus, the present results indicate that the two G(s)-coupled PACAP I and PACAP II VIP1 receptors may stimulate IP production. The maximal stimulation depended on the number of receptor expressed; the PACAP I and PACAP II VIP1 receptors probably activated the phospholipase C through G proteins of the G(q), and of the G(i)/G(o) families, respectively.

Role of phosphodiesterase II in cross talk between cGMP and cAMP in human neuroblastoma NB-OK-1 cells.

Cyclic nucleotides levels and cyclic nucleotide phosphodiesterase (PDE) activities were measured in human neuroblastoma NB-OK-1 cells possessing atrial natriuretic peptide (ANP) receptors of the A type and pituitary adenylate cyclase activating polypeptide (PACAP)-preferring receptors. Adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) degradation were interrelated since the increase in cGMP, induced by ANP-(99-126), stimulated the hydrolysis of cAMP by PDE isoenzyme II. In intact NB-OK-1 cells, the levels of cAMP and cGMP attained in the presence of, respectively, 1 nM PACAP-(1-27) and 10 nM ANP-(99-126), and in the absence or presence of PDE inhibitors, strongly suggested that cAMP hydrolysis was mainly achieved by isoenzyme IV, and to a lesser extent by isoenzymes I, II, and III, while cGMP was degraded by isoenzymes I, II, III, and V. More than one-half of total cAMP- and cGMP-hydrolyzing activities was present in the membrane-bound fraction. Cyclic nucleotide PDE activities separated by anion-exchange chromatography showed that isoenzymes III and IV were mainly present in the membrane fraction, while isoenzymes I, II, and V were in the cytosolic fraction.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide stimulate two signaling pathways in CHO cells stably transfected with the selective type I PACAP receptor.

The properties of the pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor were studied on a clone of Chinese hamster ovary cells (CHO) stably transfected with the recombinant receptor. PACAP(1-27), PACAP(1-38) and VIP inhibited [125I-acetyl-His1]PACAP (1-27) binding, stimulated cyclic AMP and inositol phosphates production and induced [Ca2+]i increase with the same order of potency: PACAP(1-27) = PACAP(1-38) > VIP. The concentrations required for half maximal receptor occupancy, IP3- and [Ca2+]i increase were not different for both PACAPs (1 nM) and 100-fold higher than those required for cyclic AMP increase (0.010 nM). These data suggest that the occupancy of a portion of the total receptors available was sufficient for maximal cyclic AMP production but not for maximal IP3 production. It is concluded that the possibility of the type I PACAP receptor being coupled to a transduction pathway is not located at the level of the ligand but rather at the level of the G-proteins.

New evidence for a membrane-bound pathway in hormone receptor binding.

The fully active cholecystokinin analog (Thr,Nle)-CCK-9 was lipo-derivatized by N-terminal grafting of a dimyristoylglycerol moiety to induce tight interdigitation with cell membrane bilayers. While the parent CCK peptide was shown to interact only transiently with small unilamellar phospholipid vesicles, the lipo-CCK peptide, although self-aggregating into vesicles, inserts rapidly and quantitatively into phospholipid bilayers. Fluorescence and, even more so, NMR data are supportive for a chain reversal of the CCK moiety of the lipo derivative with embedment of the C-terminus into hydrophobic compartments of the bilayer. MD simulations allowed for a proposal of the folded form of CCK in bilayers with a helical array parallel to the interface and an amphipathic display of the side chains. In this model, the phenylalanine aromatic ring is heading the peptide molecule and may thus play a decisive role in the lateral penetration of the receptor at the water/lipid interface. In fact, despite the membrane-bound state, its binding affinity for rat pancreatic acini is comparable to that of the CCK peptide when tested after a 3-h equilibration period but 5-6-fold lower at 45 min, suggesting that the association rate is significantly lower than that of the unmodified CCK peptide. This can rationally be attributed to the tight interdigitation of the double-tailed lipo moiety with the membrane bilayer. Moreover, an escape of the lipopeptide into the extracellular aqueous phase is energetically highly unfavored; therefore, the receptor can only be reached by a membrane-bound two-dimensional migration. The observed difference in amplification between binding and amylase secretion may result from inadequate occupation of low-affinity CCK receptors, which leads then to poor couplings to G-proteins. Nevertheless the data confirm that lateral penetration of receptor structures is possible, and thus, preadsorption of peptide (neuro)hormones at the cell membrane bilayer may indeed represent the first step in the receptor recognition process.

Contrasting effects of PACAP and carbachol on [Ca2+]i and inositol phosphates in human neuroblastoma NB-OK-1 cells.

The effects of PACAPs on [Ca2+]i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1-27) and PACAP(1-38) increased [Ca2+]i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca2+]i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca2+. Carbachol also increased [Ca2+]i in a biphasic manner, but it mobilized intracellular Ca2+ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca2+ entry, this being accompanied by a more marked and prolonged elevation of IP3 and IP4 than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca2+ handling through PACAP receptors than with muscarinic M1 receptors.

Regulation of Na-K-Cl cotransport, Na,K-adenosine triphosphatase, and Na/H exchanger in human neuroblastoma NB-OK-1 cells by atrial natriuretic peptide.

The occupancy of atrial natriuretic peptide (ANP) receptors of the ANPA type in human neuroblastoma NB-OK-1 cells elevates cGMP. In this study, ANP concentrations of 10 nM or more increased total K+ uptake. Data obtained in the presence of bumetanide and/or ouabain demonstrated that 1 microM ANP induced a primary stimulation (by 82%) of Na-K-Cl cotransport and a subsequent indirect stimulation (by 15%) of Na,K-ATPase. ANP also inhibited Na/H exchange through an amiloride-sensitive mechanism, as shown by intracellular pH measurement in cells challenged or not by an acid or alkaline load. (Bu)2cGMP mimicked all ANP effects, suggesting that ANP acted through a cGMP-dependent mechanism.

Discovery of a potent atrial natriuretic peptide antagonist for ANPA receptors in the human neuroblastoma NB-OK-1 cell line.

The effects of seven competitive atrial natriuretic peptide (ANP) receptor antagonists were compared on cultured human neuroblastoma NB-OK-1 cells expressing exclusively ANPA receptors, by evaluating their capacity to inhibit [125I]ANP binding and to suppress ANP-stimulated cyclic GMP elevation. In ANP analogues with a shortened Cys7-Cys18 bridge, Asp13 and a hydrophobic Tic residue at position 16 expressed antagonistic activity, while Ala16 provoked lower antagonistic potency and Phe16 induced receptor activation. The binding affinity of A71915 ([Arg6, Cha8]ANP-(6-15)-D-Tic-Arg-Cys-NH2), the most potent antagonist (with a pKi of 9.18 and a pA2 of 9.48) was only 22 times less lower than that of the agonist ANP-(1-28).

Atrial natriuretic peptide binds to ANP-R1 receptors in neuroblastoma cells or is degraded extracellularly at the Ser-Phe bond.

ANP-R1 receptors for atrial natriuretic peptide (ANP) showed the following rank order of affinity in intact human neuroblastoma cells NB-OK-1: human ANP-(99-126) approximately human ANP-(102-126) approximately rat ANP-(99-126) (K1 17-32 pM) > human ANP-(103-126) > porcine brain natriuretic peptide (BNP). Analogues truncated at the C-terminal extremity or devoid of a disulphide bridge, such as rat ANP-(103-123), rat C-ANP-(102-121), rat ANP-(111-126), rat ANP-(99-109) and rat [desCys105,Cys121]ANP-(104-126) and chicken C-type natriuretic peptide, were not recognized. The occupancy of these high affinity ANP-R1 receptors led to marked cyclic GMP accumulation in the presence of 3-isobutyl 1-methylxanthine. An ectoenzymic activity, partly shed in the incubation medium, provoked the stepwise release of Phe-Arg-[125I]Tyr, Arg-[125I]Tyr and [125I]Tyr from rat [125I]ANP-(99-126), at an optimal pH of 7.0. Its inhibition by 1,10-phenanthroline, EDTA and bacitracin but not by thiorphan suggests the contribution of at least one neutral metalloendopeptidase, distinct from EC 3.4.24.11, for which ANP showed high affinity.

Inhibitory effects of ATP and other nucleotides on atrial natriuretic peptide (ANP) binding to R1-type ANP receptors in human neuroblastoma NB-OK-1 cell membranes.

ATP dose-dependently inhibited rat 125I-ANP-(99-126) binding to membranes from the human neuroblastoma cell line NB-OK-1 by increasing the KD value for the hormone without altering the Bmax value. After a 20 min preincubation with 37.5 pM 125I-ANP-(99-126) and 0.5 mM ATP, followed by the addition of 0.3 microM unlabelled ANP-(99-126), the proportion of rapidly dissociating receptors was 4-times higher than in the absence of ATP. The other nucleotides ADP, AMP, AMP-PNP, ATP gamma S, GTP, GDP, GMP, GMP-PNP and GTP gamma S were also inhibitory but with a lower potency and/or efficacy. Binding equilibrium data were satisfactorily simulated by a computer program based on partially competitive binding of ANP-(99-126) and the nucleotides, and this, together with the data on dissociation kinetics, strongly suggests that several nucleotides, when added at concentrations up to 1 mM, form a ternary ANP-receptor-nucleotide complex.

A new metallo- endopeptidase from human neuroblastoma NB-OK-1 cells which inactivates atrial natriuretic peptide by selective cleavage at the Ser123-Phe124 bond.

A novel metallo-endopeptidase from human neuroblastoma NB-OK-1 cells was partially purified and characterized. This enzyme activity was detected in the culture medium and could be detached from intact cells by gentle washing, suggesting a peripheral localization of the enzyme. This endopeptidase inactivated Atrial Natriuretic Peptide (ANP) by a unique and selective cleavage of the Ser123-Phe124 bond. It also produced hydrolysis at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bonds of other peptide hormones such as bradykinin, somatostatin 14, litorin, substance P, neuromedin C and angiotensin II. The substrate selectivity and inhibition profile of the enzyme showed obvious similarities with the peptide hormone inactivating endopeptidase (PHIE) recently purified from Xenopus laevis skin secretions and indicated a thermolysin-like activity distinct from neutral endopeptidase (EC 3.4.24.11) and from angiotensin converting enzyme (EC 3.4.15.1).

Functional cholecystokinin receptors are distinguished kinetically by biotinyl-Tyr-Gly-(Thr28,Nle31)CCK(25-33) in rat pancreatic acini.

Biotinyl-tyrosine-glycine(Thr28,Nle31)CCK(25-33) (BTG-TN-CCK-9) promoted amylase secretion and phosphatidylinositol (PI) metabolism with the same potency and efficacy as TN-CCK-9 in dispersed rat pancreatic acini. A 1 min preincubation of the ligand with a 20-fold excess of streptavidin completely suppressed this biological activity. On the other hand, amylase secretion and PI metabolism prestimulated with BTG-TN-CCK-9 were blocked within 1-5 min after streptavidin addition. [125I]BTG-TN-CCK-9 bound to high (Kd 0.17 nM) and low (Kd 13 nM) affinity receptors. Its dissociation, in the presence of either streptavidin or TN-CCK-9, showed a rapid component and a slow component. The proportion of tracer dissociating slowly increased with increasing preincubation time as did the proportion of tracer that could not be washed away quickly by acidic treatment, in parallel experiments. This phenomenon occurred less readily at 4 degrees C or in the presence of 1 mM CCCP. In acini preincubated for 30 min with 0.3 nM [125I]BTG-TN-CCK-9 and various concentrations of unlabelled BTG-TN-CCK-9, then washed at neutral pH (in order to eliminate rapidly dissociating ligand preferentially), the tracer displacement curve was shifted leftward, suggesting that rapidly dissociating receptors corresponded to low affinity receptors. When acini were preincubated for 1 min with BTG-TN-CCK-9, then washed at neutral pH with buffer only, we observed residual stimulated secretion over the next 30 min period, that correlated with the BTG-TN-CCK-9 concentration offered during the short preincubation period. This phenomenon was inhibited by streptavidin suggesting that intracellularly accumulated intact BTG-TN-CCK-9 (as shown, by radio-HPLC) promoted residual secretion when free to bind again to cell surface receptors in the absence of streptavidin. Taken collectively, these data suggest the coexistence of at least 2 types (or states) of CCK receptors.

Characterization and regulation of atrial natriuretic peptide (ANP)-R1 receptors in the human neuroblastoma cell line NB-OK-1.

We characterized in membranes from the human neuroblastoma cell line NB-OK-1, an ANP-R1 receptor (Mr 130 kDa) for the atrial natriuretic peptide (ANP). This receptor recognized biologically active forms of ANP with high affinity but showed no affinity for truncated ANP forms. It was functional in that binding correlated with guanylate cyclase activation (a 2-fold increase in Vmax) with the following rank order of potency: rat ANP-(99-126) greater than human ANP-(99-126) greater than human ANP-(102-126) greater than porcine BNP (brain natriuretic peptide). The enzyme required free Mn2+ in addition to the Mn-GTP substrate (Km of about 0.3 mM for both basal and ANP-stimulated activity). In the presence of dithiothreitol, the dose-response curve of guanylate cyclase activation was shifted rightward by a factor of 30. ANP-R1 receptors were upregulated through protein synthesis in cells exposed to 1 mM carbamylcholine or 1 mM dibutyryl cyclic AMP for 8-24 h (ANP was ineffective).

The metabolic effects of guanyl nucleotides on rat pancreatic acini permeabilized with streptolysin O suggest a widespread use of G proteins.

In streptolysin O permeabilized acini that were normally responsive to carbamylcholine and cholecystokinin octapeptide, amylase secretion was stimulated: a) by the stable guanyl nucleotides with a potency decreasing as follows: guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) greater than guanylyl-imidodiphosphate (GMP-PNP) = guanylyl-(beta, gamma-methylene)-diphosphonate (GMP-PCP), in the presence of 0.5 mM calcium and b) by calcium alone (EC50 3 microM). The maximal secretory effect of calcium alone (a 2-fold increase) was less effective than that of GTP gamma S and calcium offered in combination (an 8-fold increase). In the virtual absence of Ca2+, GTP gamma S still stimulated amylase release (a 3-fold increase) while 12-O-tetradecanoylphorbol 13-acetate (TPA) did not. The relative potencies of guanyl nucleotides were GTP gamma S greater than GMP-PNP = GMP-PCP = GTP on phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown, GTP gamma S greater than GMP-PNP greater than GMP-PCP = GTP on 45Ca2+ efflux, and GTP GMP-PNP = GMP-PCP = GTP gamma S on [1-14C]arachidonate efflux. Based on these data, the contribution of G proteins to stimulus-secretion coupling beyond the transduction of receptor signal is considered.

Purification and characterization of five variants of phospholipase A2 and complete primary structure of the main phospholipase A2 variant in Heloderma suspectum (Gila monster) venom.

1. Five increasingly anionic variants (Pa1-Pa5) of Ca2+-dependent phospholipase A2 were purified to homogeneity from the venom of the lizard Heloderma suspectum (Gila monster). The purification procedure was based on semi-preparative reverse-phase HPLC followed by anion-exchange HPLC and analytical reverse-phase HPLC. 2. Their Mr were 17,000-18,000, as deduced by SDS/PAGE. Specific activities tested by the capacity to hydrolyze phosphatidylcholines at pH 8.5 decreased as follows: Pa3 greater than Pa5 greater than Pa4 greater than Pa1 greater than Pa2. These activities showed the same optimum pH (9.0), were mainly of the phospholipase A2 type and were lost upon p-bromophenacyl bromide treatment. 3. All five phospholipases efficiently stimulated amylase release from dispersed rat pancreatic acini at pH 7.4, their potency decreasing as follows: Pa2 greater than Pa1 approximately equal to Pa4 greater than Pa3 approximately equal to Pa5. No deleterious effect was apparent based on the lack of lactate dehydrogenase release. 4. The five variants, Pa1-Pa5, differed significantly in amino acid composition and this, together with distinct antigenic properties of Pa2 and Pa5, establishes the subheterogeneity of this new type of phospholipase A2, despite the fact that the N-terminal amino acid sequence (31 residues) of Pa1-Pa5 was exactly the same. 5. The full sequence of the major variant, Pa5, showed that this 142-amino-acid protein exhibited greater similarity to the bee venom enzyme than to any class I or class II secretory phospholipase A2 from snake venom and mammalian pancreas. While Pa5 displayed the highly conserved region between Asp30 and Cys39 (the essential active site of all phospholipases A2), its salient original points included 10 half-cystine residues only, an incomplete N-terminal sequence, large changes in the putative calcium loop, several alterations after the active site and a C-terminal extension never seen in other phospholipases A2, with the only exception being bee venom.

Functional characterization of muscarinic receptors in rat parotid acini.

The muscarinic agonist, carbamylcholine, stimulated amylase secretion in rat parotid acini 6-fold, the 86Rb efflux 5-fold, the 45Ca efflux 5-fold and the accumulation of inositol monophosphate, bisphosphate, trisphosphate and tetrakisphosphate 4-, 4-, 3- and 3-fold, respectively. The EC50 of carbamylcholine on these parameters were 0.4, 0.5, 1.3, 12, 12, 6 and 9 microM, suggesting spareness between phospholipase C activation and amylase secretion. These muscarinic responses were inhibited by four muscarinic antagonists with an order of potency on all parameters and on receptor occupancy (using N-[methyl-3H]scopolamine as a tracer): atropine greater than hexahydrosiladifenidol greater than pirenzepine greater than AF-DX 116. The pA2 of these antagonists on carbamylcholine-stimulated amylase secretion were 9.72 for atropine, 8.14 for hexahydrosiladifenidol, 7.16 for pirenzepine and 6.22 for AF-DX 116, indicating that the parotid muscarinic receptors were of an M2 subtype 83-fold more sensitive to hexahydrosiladifenidol than to AF-DX 116.