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.

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.

Insulinotropic effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in rat pancreatic islets.

In isolated rat pancreatic islets, the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), when used in the 2.10(-9) to 2.10(-7) M range, was found to stimulate insulin release both in the absence and presence of glucose. The non-tumor-promoting agent 4-methylphorbol-12,13-didecanoate failed to stimulate insulin release. The insulinotropic capacity of TPA was enhanced by glucose in a dose-related fashion. In the absence of glucose, the TPA-stimulated release of insulin was a slowly induced and not rapidly reversible phenomenon. It was inhibited by antimycin A, by epinephrine, at low temperatures, and in the absence of extracellular Ca2+ or the presence of cytochalasin B, was unaffected by the organic calcium antagonist D600 or indomethacin, and was potentiated by theophylline. No obvious effect of TPA upon 86Rb or 32P efflux and 45Ca net uptake could be detected in the isolated islets. However, TPA caused a progressive increase in both 45Ca fractional outflow rate and cyclic adenosine 3':5'-monophosphate content in the islets. It is proposed that the insulinotropic action of TPA may be due, in part at least, to interference with the transport of calcium by native ionophores.

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.

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.

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.

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.

Scorpion venom inhibits carbamylcholine-induced 86rubidium efflux from rat parotid acini.

The muscarinic agonist carbamylcholine stimulated 5-fold 86Rb efflux from preloaded rat parotid acini. Apamin was without effect on this carbamylcholine-induced 86Rb efflux. By contrast, the venom from Leiurus quinquestriatus (a scorpion from Israel) inhibited non-competitively this efflux while being without effect on the carbamylcholine-stimulated 45Ca efflux and amylase release. This heat-resistant inhibitory effect of the venom was destroyed by boiling in the presence of dithiothreitol. These results suggest that the venom from L. quinquestriatus contains a toxin capable to block apamin-insensitive calcium-activated potassium channels in rat parotid acini.

Purification of a novel pancreatic secretory factor (PSF) and a novel peptide with VIP- and secretin-like properties (helodermin) from Gila monster venom.

A combination of three HPLC procedures applied to the venom of Gila monster (Heloderma suspectum) has led to the purification to homogeneity of two bioactive components: (i) a 17.5 kDa protein, isolated on the basis of its potent secretory effect on dispersed rat pancreatic acini, was accordingly designated PSF (pancreatic secretory factor); (ii) a 5.9-kDa peptide, designated helodermin, was purified on the basis of its ability to stimulate adenylate cyclase in rat pancreatic membranes. PSF was unable to activate adenylate cyclase and, conversely, helodermin was devoid of secretory action.

Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties.

Helodermin, a newly isolated peptide from the venom of Gila monster (Heloderma suspectum) was shown to stimulate the adenylate cyclase activity of rat pancreatic membranes as efficiently as secretin and VIP. It also increased cyclic AMP levels and inhibited [125I]VIP binding in rat pancreatic acini. Finally, helodermin activated adenylate cyclase in membranes from rat heart, rat brain, and human heart, showing properties analogous yet distinct from those of secretin, VIP and PHI.

Pancreatic secretory factor (PSF), a protein from Gila monster venom stimulating enzyme secretion from rat pancreatic acini.

Pancreatic secretory factor (PSF), a 17.5-kDa protein purified from the venom of Gila monster (Heloderma suspectum), stimulated amylase secretion from dispersed rat pancreatic acini more efficiently than CCK-8, bombesin, carbachol and secretin, and without increasing 45Ca2+ efflux and cyclic AMP levels. The secretory action was dependent on the presence of extracellular calcium and was additive to the secretion induced by agents acting via cyclic AMP or via Ca2+ efflux.

Phospholipase A2 activity of pancreatic secretory factor, a new secretagogue isolated from the venom of Heloderma suspectum.

Pancreatic secretory factor (PSF), an efficient pancreatic secretagogue recently isolated from the venom of Heloderma suspectum, is shown to exert phospholipase A2 activity towards phosphatidylcholine. This activity is strictly dependent on calcium (apparent Ka 40 nM) and has an optimum pH around 9. At pH 7.4 and in the presence of calcium, PSF retains 40% of its phospholipase A2 activity. These results are compared to the calcium dependency of the secretory effect of PSF on rat pancreatic acini. Taken collectively, the present data on PSF suggest that a similar endogenous phospholipase A2 activity might be involved in the late steps of stimulus-secretion coupling in the exocrine pancreas.

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.

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.

Vasoactive intestinal peptide receptors in pancreas and liver. Structure-function relationship.

In purified rat pancreatic plasma membranes, (D-Phe4)PHI interacts as a selective VIP agonist for rat pancreatic VIP-preferring receptors, based on binding selectivity and adenylate cyclase activation, therefore allowing us to discriminate between the participation of VIP-preferring and secretin-preferring receptors in VIP stimulation. VIP-preferring receptors also bind GRF. They rely on disulfide bridges for their functional integrity. Their coupling with adenylate cyclase, based on the intrinsic activity of VIP analogues, is poor when compared to that of hepatic VIP receptors. In fresh rat liver plasma membranes, high-affinity VIP receptors are specifically labeled with [125I]helodermin and [125I]His1, D-Ala NLeu27)GRF and are well coupled to adenylate cyclase while low-affinity VIP receptors are not. The first subtype of VIP receptors is highly responsive to guanyl nucleotides and is easily altered by dithiothreitol. Only after freezing and thawing are low-affinity hepatic VIP receptors coupled to adenylate cyclase. Concerning the chemical characterization of VIP receptors, 66- and 35-kDa peptides are detected after specific [125I]VIP cross-linking with double agents in rat pancreatic membranes. In contrast, in intact pancreatic acini, the main source of radioactivity has a molecular mass of 130-180 kDa (with no contribution of intramolecular disulfide bridges), and an 80-kDa peptide is also detectable. The 66-kDa species in membranes can conceivably derive from the 80-kDa species observed in intact cells. Its molecular mass is higher than that of the 56-kDa [125I]VIP cross-linked protein previously observed in rat liver membranes. Besides, species differences between rat and guinea pig pancreas are also evident.

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.

Stimulatory effects of Gila monster venom on rat pancreatic acini.

The effects of Gila monster venom on dispersed rat pancreatic acini were compared with those of secretin and VIP. The efficacy of the venom in terms of amylase release was much higher (a 24-fold increase over basal secretion) than that of secretin (a 4-fold increase) and VIP (+ 40% only). On the other hand, cyclic AMP levels increased 12-fold with the venom, as compared to 18-fold with secretin and 16-fold with VIP. The venom, VIP and secretin all displaced 125I-VIP and the competition curve with the venom was steeper, suggesting that all VIP-recognizing receptors bound the venom with the same affinity. VIP receptors were, however, not responsible for the release of amylase provoked by the venom since VIP (and secretin) did not inhibit the secretory action of the venom. The venom exerted no effect on 45Ca efflux and its secretory effect did not depend on the presence of external calcium. Besides, the effect of CCK-8 on amylase release was additive with the effect of the venom. A first exposure to the venom induced a refractoriness to itself with respect to amylase release but not in terms of cyclic AMP increase. In conclusion, Gila monster venom may contain one component binding to VIP/secretin receptors with resulting cyclic AMP elevation. A second venom component may be responsible for the high secretory efficacy, without involving cyclic AMP or calcium efflux.

Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8).

Rat pancreatic acini were preincubated with 0.4 mM 32Pi for 45 min at 37 degrees C, then exposed for 15 min to VIP, secretin or CCK-8. The incubation was terminated with a stop solution and a fraction rich in mitochondria and zymogen granules was separated from a microsome-rich fraction by differential centrifugation. After heating in the presence of SDS, beta-mercaptoethanol was added and the pattern of equivalent amounts of 32P-labelled proteins was examined by autoradiography of SDS-PAGE gels. VIP, secretin, and CCK-8 stimulated the phosphorylation of a Mr=33 K microsomal protein and that of two proteins of Mr=21 K and Mr=25 K mostly present in a fraction rich in mitochondria and zymogen granules. Stimulations were dose-dependent, the highest stimulant concentrations tested allowing 2- to 3-fold increases of phosphorylation over basal. When 1 nM CCK-8 was used simultaneously with 1 microM VIP, the cyclic AMP levels attained and the pattern of protein phosphorylation were similar to those obtained with VIP alone, and there was a potentiation of amylase secretion; when a supra-maximal 0.1 microM CCK-8 concentration was added, the VIP-induced elevation in cyclic AMP levels and the phosphorylation of the Mr=21 K and Mr=25 K proteins were partially antagonized, and no potentiation any more of secretion occurred. To conclude the in vitro phosphorylation of three particulate proteins (Mr=33 K, 25 K, and 21 K) was similarly increased in rat pancreatic acini in response to secretin and VIP (acting through cyclic AMP) and to CCK-8 (acting mostly through Ca2+).

Effector mechanisms of peptides of the VIP family.

The present review is focused on the exocrine pancreas and liver where the only known effector mechanism of VIP is the activation of adenylate cyclase in plasma membranes. A two-state model of activation-deactivation of the enzyme visualizes the participation of VIP receptors and Ns, the guanyl nucleotide stimulatory protein of adenylate cyclase. In the rat pancreas, VIP and GRF receptors are indistinguishable and disulfide bridges influence their functional integrity. The antagonism of VIP and somatostatin perhaps requires, at the adenylate cyclase level, the contribution of Ni, the guanyl nucleotide inhibitory protein. The potentiation of VIP by various stimulants acting on Ca2+ movements may rely on later events, e.g., on a concerted activation of protein kinases. When comparing quantitatively peptide binding to receptors with adenylate cyclase activation, cyclic AMP levels and amylase secretion, a tool is at hand to tailor synthetic agonists and antagonists of VIP, with appropriate changes in the N-terminal moiety of the peptide (a good agonist allows efficient coupling of receptors to the adenylate cyclase system). Apart from stimulus-secretion coupling, VIP may influence protein synthesis in the rat pancreas, through the phosphorylation of ribosomal protein S6, and may alter the activity of the endoplasmic reticulum via the phosphorylation of Mr = 21 kDa and Mr = 25 kDa proteins. In rat liver membranes, high affinity VIP receptors are specifically labelled with 125I-helodermin and are coupled to adenylate cyclase (at variance with low affinity VIP receptors). These receptors are highly responsive to divalent cations and to guanyl nucleotides.

Adenylate cyclase stimulation by VIP in rat and human parotid membranes.

Adenylate cyclase activity was stimulated by vasoactive intestinal peptide (VIP) in rat parotid membranes, in the presence of 100 microM guanosine triphosphate (GTP). The threshold concentration of VIP was 300 nM and the activity doubled at the maximal VIP concentration tested (30 microM). The relative potency of peptides of the VIP family was: VIP greater than peptide histidine isoleucinamide (PHI) greater than secretin. The beta-adrenergic agent isoproterenol was a more efficient activator of rat parotid adenylate cyclase and its stimulatory effect, like that of VIP, depended on the presence of GTP. The effects of VIP and isoproterenol were both potentiated by 10 microM forskolin. By comparison with rat parotid preparations, membranes from a human parotid gland responded similarly to the VIP family of peptides (VIP greater than PHI greater than secretin). In both rat and human parotid membranes, two proteins (Mr 44 kDa and 53 kDa) of the alpha-subunit of Ns (the guanyl nucleotide-binding stimulatory protein) were labelled by ADP-ribosylation, in the presence of cholera toxin. Taken together, these results indicate that VIP receptors, when coupled to Ns, were able to activate the adenylate cyclase system in rat and human parotid membranes.