Influence of organizational stress on reported depressive symptoms among police.

BACKGROUND: There is a growing body of research on operational stress injuries (OSIs) among police officers and first responders. Most studies focus on operational stressors' contribution to OSI and the development of post-traumatic stress disorder. However, preliminary research shows that organizational stressors may uniquely contribute to OSI and depression, and thus should be examined more closely. AIMS: This study explored the influence of organizational stress on symptoms of depression in a sample of police officers from a large urban region. METHODS: Front-line (n = 109) police officers completed questionnaires measuring police organizational and operational stress, depression, anxiety, hostility, rumination, perceived social support and social desirability. Using negative binomial regression (NBR), a best subset model of self-reported depression symptoms was derived from the full model (a function of gender, age, police experience (years), organizational stress, operational stress, anxiety, anger, rumination and social support), based on Akaike Information Criterion (AIC) goodness of fit. RESULTS: Organizational stress and anxiety were positively associated with self-reported depression symptoms. A paired t-test revealed no significant difference between reported organizational and operational stress levels. CONCLUSIONS: Organizational stress may uniquely contribute to OSI and depressive symptoms and should be examined in future research. Findings support prior literature suggesting that initiatives to treat OSI among police should address workplace environment and organizational stressors. Addressing organizational issues in police culture and developing long-lasting initiatives is key in the future of OSI prevention and treatment for police officers.

The Impact of Acute Stress Physiology on Skilled Motor Performance: Implications for Policing.

Investigations of police performance during acutely stressful situations have primarily focused on higher-order cognitive processes like attention, affect or emotion and decision-making, and the behavioral outcomes of these processes, such as errors in lethal force. However, behavioral outcomes in policing must be understood as a combination of both higher-order processes and the physical execution of motor skills. What is missing from extant police literature is an understanding of how physiological responses to acute stress contribute to observed decrements in skilled motor performance at the neuromuscular level. The purpose of the current paper is to fill this knowledge gap in the following ways: (1) review scientific evidence for the physiological (i.e., autonomic, endocrine, and musculoskeletal) responses to acutely stressful exposures and their influence on skilled motor performance in both human and animal models, (2) review applied evidence on occupationally relevant stress physiology and observed motor decrements in performance among police, and (3) discuss the implications of stress physiology for police training and identify future directions for applied researchers. Evidence is compelling that skill decay is inevitable under high levels of acute stress; however, robust evidence-informed training practices can help mitigate this decay and contribute to officer safety.

ATP binding residues of sarcoplasmic reticulum Ca2+-ATPase.

ATP-binding residues in the N and P domains of sarcoplasmic reticulum Ca-ATPase have been investigated using mutagenesis in combination with a binding assay based on the photolabeling of Lys(492) with [g-(32)P] 2',3'-O-(2,4,6 trinitrophenyl)-8-azido-ATP and competition with nucleotide. In the N domain, mutations to several residues in conserved motifs, (438)GEATE, (487)FSRDRK, (515)KGAPE, and (560)RCLALA produce nucleotide-binding defects. Key residues include Thr(441), Glu(442), Phe(487), Arg(489), Lys(492), Lys(515), Arg(560), and Leu(562). In the absence of Mg(2+), Arg(489), Lys(492), and Arg(560) are most important, whereas in its presence Thr(441) and Glu(442) also play a crucial role. In the P domain, Asp(351) is striking for its strong electrostatic repulsion of the gamma-phosphate, especially in the presence of Mg(2+). Lys(352) is a key residue, and Asp(627) and Lys(684) must come close to the nucleotide. Thr(353), Asn(359), Asp(601), and Asp(703) interact only in the presence of Mg(2+). Asn(706) and Asp(707) are unimportant for nucleotide binding. The results identify several ATP binding residues in the N and P domains and suggest that Mg(2+) changes the nucleotide/protein interaction in both. Models of bound ATP and MgATP are presented.

Importance of Thr-353 of the conserved phosphorylation loop of the sarcoplasmic reticulum Ca2+-ATPase in MgATP binding and catalytic activity.

Mutants in which Thr-353 of the Ca(2+)-ATPase of sarcoplasmic reticulum had been replaced with alanine, serine, glutamine, cysteine, valine, aspartate, or tyrosine were analyzed functionally. All the mutations severely affected MgATP binding, whereas ATP binding was close to normal in the alanine, serine, glutamine, and valine mutants. In the serine and valine mutants, the maximum rate of phosphorylation from MgATP was 8- and 600-fold lower, respectively, compared with wild type. Replacement of Mg(2+) with Mn(2+) led to a 1.5-fold enhancement of the maximum phosphorylation rate in the valine mutant and a 5-fold reduction in the wild type. The turnover of the phosphoenzyme formed from MgATP was slowed 1-2 orders of magnitude relative to wild type in the alanine, serine, and valine mutants, but was close to normal in the aspartate and cysteine mutants. Only the serine mutant formed a phosphoenzyme in the backward reaction with P(i), and the hydrolysis of this intermediate was greatly enhanced. Analysis of the functional changes in the mutants in the light of the recent high resolution structure of the Ca(2+)-ATPase crystallized without the MgATP substrate suggests that, in the native activated state of the enzyme, the side chain hydroxyl of Thr-353 participates in important interactions with nucleotide and phosphate, possibly in catalysis, whereas the main chain carbonyl of Thr-353, but not the side chain, may coordinate the catalytic Mg(2+).

Importance of transmembrane segment M3 of the sarcoplasmic reticulum Ca2+-ATPase for control of the gateway to the Ca2+ sites.

The specific functional roles of various parts of the third transmembrane segment (M3) of the sarcoplasmic reticulum Ca(2+)-ATPase were examined by functionally characterizing a series of mutants with multiple or single substitutions of M3 residues. Steady-state and transient kinetic measurements, assisted by computer simulation of the time and Ca(2+) dependences of the phosphorylation level, were used to study the partial reaction steps of the enzyme cycle, including the binding and dissociation of Ca(2+) at the high affinity cytoplasmically facing sites. The mutation Lys-Leu-Asp-Glu(255) --> Glu-Ile-Glu-His resulted in a conspicuous increase in the rate of Ca(2+) dissociation as well as a displacement of the major conformational equilibria of the phosphoenzyme and dephosphoenzyme forms. The point mutant Phe(256) --> Ala also showed an increased rate of Ca(2+) dissociation, whereas a conspicuous decrease both in the rate of Ca(2+) dissociation and in the rate of Ca(2+) binding was found for the mutant Gly-Glu-Gln-Leu(260) --> Ile-His-Leu-Ile. These findings suggest that the NH(2)-terminal half of M3 is involved in control of the gateway to the Ca(2+) sites. The main effect of two mutations to the COOH-terminal half of M3, Ser-Lys-Val-Ile-Ser(265) --> Thr-Gly-Val-Ala-Val and Leu-Ile-Cys-Val-Ala-Val-Trp-Leu-Ile(274) --> Phe-Leu-Gly-Val-Ser-Phe-Phe-Ile-Leu, was a block of the dephosphorylation.

Importance of stalk segment S5 for intramolecular communication in the sarcoplasmic reticulum Ca2+-ATPase.

Sixteen residues in stalk segment S5 of the Ca(2+)-ATPase of sarcoplasmic reticulum were studied by site-directed mutagenesis. The rate of the Ca(2+) binding transition, determined at 0 degrees C, was enhanced relative to wild type in mutants Ile(743) --> Ala, Val(747) --> Ala, Glu(748) --> Ala, Glu(749) --> Ala, Met(757) --> Gly, and Gln(759) --> Ala and reduced in mutants Asp(737) --> Ala, Asp(738) --> Ala, Ala(752) --> Leu, and Tyr(754) --> Ala. In mutant Arg(762) --> Ile, the rate of the Ca(2+) binding transition was wild type like at 0 degrees C, whereas it was 3.5-fold reduced relative to wild type at 25 degrees C. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was increased conspicuously in mutants Ile(743) --> Ala and Tyr(754) --> Ala (close to 20-fold in the absence of K(+)) and increased to a lesser extent in Asn(739) --> Ala, Glu(749) --> Ala, Gly(750) --> Ala, Ala(752) --> Gly, Met(757) --> Gly, and Arg(762) --> Ile, whereas it was reduced in mutants Asp(737) --> Ala, Val(744) --> Gly, Val(744) --> Ala, Val(747) --> Ala, and Ala(752) --> Leu. In mutants Ile(743) --> Ala, Tyr(754) --> Ala, and Arg(762) --> Ile, the apparent affinities for vanadate were enhanced 23-, 30-, and 18-fold, respectively, relative to wild type. The rate of Ca(2+) dissociation was 11-fold increased in Gly(750) --> Ala and 2-fold reduced in Val(747) --> Ala. Mutants with alterations to Arg(751) either were not expressed at a significant level or were completely nonfunctional. The findings show that S5 plays a crucial role in mediating communication between the Ca(2+) binding pocket and the catalytic domain and that Arg(751) is important for both structural and functional integrity of the enzyme.

Fast kinetic analysis of conformational changes in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Rapid quench experiments at 25 degrees C were carried out on selected mutants of the sarco(endo)plasmic reticulum Ca(2+)-ATPase to assess the kinetics of the conformational changes of the dephosphoenzyme associated with ATP binding/phosphoryl transfer and the binding and dissociation of Ca(2+) at the cytoplasmically facing transport sites. The mutants Gly(233) --> Glu, Gly(233) --> Val, Pro(312) --> Ala, Leu(319) --> Arg, and Lys(684) --> Arg differed conspicuously with respect to the behavior of the dephosphoenzyme, although they were previously shown to display a common block of the transformation of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form. The maximum rate of the ATP binding/phosphoryl transfer reaction was reduced 3.6-fold in mutant Gly(233) --> Glu and more than 50-fold in mutant Lys(684) --> Arg, relative to wild type. In mutant Leu(319) --> Arg, the rate of the Ca(2+)-binding transition was reduced as much as 10-30-fold depending on the presence of ATP. In mutants Gly(233) --> Glu, Gly(233) --> Val, and Pro(312) --> Ala, the rate of the Ca(2+)-binding transition was increased at least 2-3-fold at acid pH but not significantly at neutral pH, suggesting a destabilization of the protonated form. The rate of Ca(2+) dissociation was reduced 12-fold in mutant Pro(312) --> Ala and 3.5-fold in Leu(319) --> Arg, and increased at least 4-fold in a mutant in which the putative Ca(2+) liganding residue Glu(309) was replaced by aspartate. The data support a model in which Pro(312) and Leu(319) are closely associated with the cation binding pocket, Gly(233) is part of a long-range signal transmission pathway between the ion-binding sites and the catalytic site, and Lys(684) is an essential catalytic residue that may function in the same way as its counterpart in the soluble hydrolases belonging to the haloacid dehalogenase superfamily.

Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.

The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.

Structure-function relationships of the calcium binding sites of the sarcoplasmic reticulum Ca(2+)-ATPase.

Site-directed mutagenesis studies of the structure and function of the Ca2+ binding sites of the sarcoplasmic reticulum Ca(2+)-ATPase are reviewed. The Ca2+ binding properties of six mutants with alterations to amino acid residues with oxygen-containing side chains in the membrane segments M4, M5, M6, and M8 were investigated. The mutations to Glu309 in M4, Glu771 in M5, Asn796, Thr799, and Asp800 in M6 all disrupted Ca2+ occlusion, suggesting that the side chains of these residues donate oxygen ligands to Ca2+ binding at the high-affinity sites and/or are involved in conformational changes that occlude the sites. Alanine substitution of Glu908 in transmembrane segment M8 did not prevent Ca2+ occlusion, thereby excluding this residue from playing a central role in Ca2+ coordination. Titrations of Ca2+ activation of phosphorylation from ATP and of inhibition by Ca2+ of phosphorylation from Pi allowed us to assign Ca2+ liganding residues separately to the two high-affinity Ca2+ sites. Hence, residues Glu771 and Thr799 are associated with the site binding the first calcium ion in the sequential mechanism ("site 1"), whereas Glu309 and Asn796 are associated with the site binding the second calcium ion ("site 2"), and Asp800 donates Ca2+ ligands to both sites. On this basis we discuss two possible structural models for the Ca2+ sites.

Mutation to the glutamate in the fourth membrane segment of Na+,K+-ATPase and Ca2+-ATPase affects cation binding from both sides of the membrane and destabilizes the occluded enzyme forms.

The functional consequences of mutations Glu329 --> Gln in the Na+,K+-ATPase and Glu309 --> Asp in the sarco(endo)plasmic reticulum Ca2+-ATPase were analyzed and compared. Relative to the wild-type Na+,K+-ATPase, the Glu329 --> Gln mutant exhibited a 20-fold reduction in the apparent K+ affinity determined by titration of the rate of ATP hydrolysis at 50 microM ATP, and the rate of release of occluded K+ or Rb+ to the cytoplasmic side of the membrane was up to 30-fold enhanced by the mutation, as measured in kinetic studies of the phosphorylation by ATP of enzyme equilibrated with K+ or Rb+. The apparent affinity for extracellular K+ was 12-fold reduced by the Glu329 --> Gln mutation, as determined by K+ titration of the dephosphorylation. The maximum rate of phosphorylation by ATP of the Na+ form of the enzyme was reduced more than 2-fold by the mutation, but this effect could be counteracted by stabilizing Na+ occlusion with oligomycin. Similar studies on the Glu309 --> Asp mutant of the Ca2+-ATPase showed that the maximum rate of phosphorylation of the Ca2+ form was 8-9-fold reduced relative to that of the wild-type Ca2+-ATPase, and no Ca2+ occlusion could be detected in the mutant. Dephosphorylation of the phosphoenzyme intermediate formed with Pi was blocked in the Ca2+-ATPase mutant. The sensitivity to inhibition by thapsigargin, which binds selectively to the putative proton-occluded form of the Ca2+-ATPase, was reduced almost 300-fold in the mutant at neutral pH, but only 3-4-fold at pH 6.0. These data indicate that the mutations destabilize the occluded enzyme forms and interfere with cation binding from the extracytoplasmic side as well as with the gating process at the cytoplasmic entrance to the cation occlusion pocket.

Mutagenesis of Sarcoplasmic Reticulum Ca(2+)-ATPase.

Ca(2+)-ATPases of sarco(endo)plasmic reticulum utilize energy derived from hydrolysis of ATP to drive active uptake of calcium ions. Site-directed mutagenesis analysis of Ca(2+)-ATPase structure-function relationships has identified protein domains and single amino acid residues involved in the binding and occlusion of the calcium ions during translocation, as well as amino acid residues crucial to the energy-transducing intramolecular signaling that links events in the catalytic ATP hydrolysis site with rearrangements in the calcium sites. The interaction between the cardiac muscle sarcoplasmic reticulum Ca(2+)-ATPase and the regulatory protein phospholamban, which mediates the activation of Ca(2+) transport by ß-agonists, has also been elucidated by mutagenesis.

Mutation Lys758 --> Ile of the sarcoplasmic reticulum Ca2+-ATPase enhances dephosphorylation of E2P and inhibits the E2 to E1Ca2 transition.

The highly conserved lysine residue Lys758 in the fifth stalk segment of the sarcoplasmic reticulum Ca2+-ATPase was substituted with either isoleucine or arginine by site-directed mutagenesis. The substitution with arginine was without significant effects on Ca2+-ATPase function, whereas multiple changes of functional characteristics were observed with the Lys758 --> Ile mutant. These included insensitivity of ATPase activity to the calcium ionophore A23187, an alkaline shift of the pH dependence of ATPase activity, reduced maximum molecular turnover rate and steady-state phosphorylation level, reduced apparent affinities for Ca2+ and inorganic phosphate, as well as increased sensitivity to inhibition by vanadate. Analysis of the partial reaction steps of the enzyme cycle traced these changes to two steps. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate (E2P) was increased, irrespective of variations of pH, K+, Ca2+, and dimethyl sulfoxide concentration. In addition, the rate of conversion of the dephosphoenzyme with low Ca2+ affinity (E2) to the Ca2+-bound form activated for phosphorylation (E1Ca2) was reduced in the mutant, and the ATP-induced rate enhancement of this step required higher ATP concentrations in the mutant compared with the wild type.

Mutagenesis of segment 487Phe-Ser-Arg-Asp-Arg-Lys492 of sarcoplasmic reticulum Ca2+-ATPase produces pumps defective in ATP binding.

The lysine residue Lys492 located in the large cytoplasmic domain of sarcoplasmic reticulum Ca2+-ATPase is implicated in nucleotide binding through affinity labeling. The contribution of segment 487Phe-Ser-Arg-Asp-Arg-Lys492 to ATP binding and pump function has been investigated through the introduction of 11 site-directed amino acid mutations. ATP binding was measured through competitive inhibition of [gamma-32P]2',3'-O-(2,4, 6-trinitrophenyl)-8-azido-adenosine triphosphate photolabeling of Lys492 or its substitute. Mutations F487S and positional swap F487S/S488F produced pumps that were severely defective in ATP binding (KD > 1 mM), and mutant F487S, together with F487E, exhibited low ATPase activity and low ATP-supported calcium transport and phosphorylation and failed to show CrATP-dependent Ca2+ occlusion. Mutations F487L, R489L, and K492Y were less inhibitory to ATP binding (KD = 8-49 microM) and, together with K492L and R489D/D490R, produced correspondingly smaller changes in ATP-mediated activities. The ATP dependence of ATPase activity of these five mutants showed deviations from the wild-type profile in the low, intermediate, and high concentration ranges, suggesting defects in ATP-dependent conformational changes. Mutations S488A and D490A had no effect on ATP binding (KD = 0.4 microM) or ATP-mediated activities. None of the mutations significantly affected phosphorylation from Pi or acetyl phosphate-supported Ca2+ transport. Mutations F487L and F487S, and not those at residue 492, increased the K0.5 for Ca2+ activation of transport 2- and 8-fold, respectively. The results implicate Phe487, Arg489, and Lys492 in binding ATP in both a catalytic and a regulatory mode.

Site-directed mutagenesis studies of energy coupling in the sarcoplasmic reticulum Ca(2+)-ATPase.

Site-directed mutagenesis studies identifying residues important to energy transduction in the sarcoplasmic reticulum Ca(2+)-ATPase are reviewed. Mutations blocking the crucial E1P to E2P transition are located in the small and the large cytoplasmic domains, in the stalk segment S4 linking transmembrane segment M4 with the catalytic site, as well as in transmembrane segments M4 and M8. Mutations that block the dephosphorylation of the E2P phosphoenzyme intermediate are located in transmembrane segments M4, M5, and M6, i.e., in the same domain as the Ca(2+)-binding sites. Removal of the sidechain of Tyr763 located at the boundary between transmembrane segment M5 and the corresponding stalk segment S5 linking M5 with the catalytic site leads to uncoupling of ATP hydrolysis from Ca2+ uptake. Uncoupling may be due to efflux through the Ca(2+)-ATPase of Ca2+ that has been transported, and may thus be caused by a defective gating process in the late part of the catalytic cycle. A nearby located residue Lys758 is also involved in energy coupling, since its substitution with Ile activates dephosphorylation at high pH and slows the E2 to E1 transition.

Dissection of the functional domains of the sarcoplasmic reticulum Ca(2+)-ATPase by site-directed mutagenesis.

The results of site-directed mutagenesis studies of the sarcoplasmic reticulum Ca(2+)-ATPase are reviewed. More than 250 different point mutants have been expressed in cell culture and analysed by a panel of functional assays. Thereby, 40-50 important amino acid residues have been pinpointed, and the mutants have been assigned to functional classes: the Ca(2+)-affinity mutants, the phosphorylation-negative mutants, the ATP-affinity mutants, the E1P mutants, the E2P mutants, and the uncoupled mutants. Moreover, regions important to the specific inhibition by thapsigargin have been identified by analysis of Ca(2+)-ATPase/Na+,K(+)-ATPase chimeric constructs.

Structure-function relationships of cation translocation by Ca(2+)- and Na+, K(+)-ATPases studied by site-directed mutagenesis.

Site-directed mutagenesis studies of the sarcoplasmic reticulum Ca(2+)-ATPase have pinpointed five amino acid residues that are essential to Ca2+ occlusion, and these residues have been assigned to different parts of a Ca2+ binding pocket with channel-like structure. Three of the homologous Na+, K(+)-ATPase residues have been shown to be important for binding of cytoplasmic Na+ at transport sites. In addition, three of the above mentioned Ca(2+)-ATPase residues appear to participate in the countertransport of H+, and two of the Na+, K(+)-ATPase residues to participate in the countertransport of K+. Residues involved in energy transducing conformational changes have also been identified by mutagenesis. In the Ca(2+)-ATPase, ATP hydrolysis is uncoupled from Ca2+ transport following mutation of a tyrosine residue located at the top of transmembrane segment M5. This tyrosine, present also in the Na+, K(+)-ATPase, may play a critical role in closing the gate to a transmembrane channel.

Functional consequences of alterations to amino acids at the M5S5 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum. Mutation Tyr763-->Gly uncouples ATP hydrolysis from Ca2+ transport.

The roles of the hydrophobic side chains of residues Phe760, Ile761, Tyr763, Leu764, and Ile765 located at the M5S5 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum were analyzed by site-directed mutagenesis. Substitution of Tyr763 with glycine resulted in a new phenotypic variant of the Ca(2+)-ATPase that catalyzed a high rate of Ca(2+)-activated ATP hydrolysis without net accumulation of Ca2+ in the microsomal vesicles. The ATPase activity of the Tyr763-->Gly mutant displayed characteristics similar to the ATPase activity of the wild-type enzyme measured in the presence of calcium ionophore, and the mutant was able to form the ADP-insensitive phosphoenzyme intermediate. Mutants Phe760-->Gly, Ile761-->Gly, Leu764-->Gly, and Ile765-->Gly were able to accumulate Ca2+. In mutants Leu764-->Gly and Ile765-->Gly, the turnover rate was low due to inhibition of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate. On the other hand, mutant Leu764-->Lys dephosphorylated rapidly. Mutants Phe760-->Gly and Leu764-->Lys displayed apparent Ca2+ affinities that were reduced two and three orders of magnitude, respectively, relative to that of the wild-type.

Nocturnal enuresis: change of nocturnal voiding pattern during alarm treatment.

In a prospective clinical study of the outcome of alarm treatment in nocturnal enuretics, 60 children were included: 40 boys and 20 girls, mean age 8.2 years (range 5.1-14.4). All were treated with enuresis alarms and had 2 or more enuretic events during the initial 14 days of treatment. None had diurnal enuresis. In each child, the enuretic and voluntary voiding frequencies during the initial 14 and last 14 days of treatment were compared. We found that 43 children had a 75% reduction or more of the enuretic events. 28 children substituted the former enuretic events by sleep, 15 changed the enuresis by voluntary voidings. Only 17 children had no effect of the alarm treatment. No parameters were found to predict the outcome. In conclusion, the outcome of successful alarm treatment occurs in two distinct forms. Either the child is left asleep without wetting his bed; or the child wakes up spontaneously from sleep and goes to the bathroom.