Does it sometimes seem that there are more coffee bars on every corner these days than pubs and service stations combined? What used to be a wake-up mug of java has now been transmogrified into an array of designer coffees - from cappuccinos to lattes to mochas to espressos to doppio espressos.

Aficionados of the coffee cult worship it as a harmless beverage, not to be consumed in excess by pregnant mothers. Its active ingredient, caffeine, is recognized as a benign psychostimulant. "Caffeine has been imbibed since ancient times in tea and coffee, and more recently in colas," observed neuroscientist Gilberto Fisone, at the Karolinska Institute in Stockholm, Sweden. "Caffeine," he writes, "owes its psychostimulant action to a blockade of adenosine A2A receptors, but little is known about its intracellular mechanism of action." He is busy filling that information gap.

Fisone is senior author of a paper in last week's issue of Nature, dated Aug. 15, 2002, and titled "Involvement of DARPP-32 phosphorylation in the stimulant action of caffeine." Its co-senior author is Nobelist Paul Greengard, who holds an endowed chair at Rockefeller University in New York, where he directs the Laboratory of Molecular and Cellular Neuroscience.

Fisone told BioWorld Today, "Our paper presents a finding that is a molecular explanation for the ability of caffeine to work as a stimulant over a period of hours - a long period of time - instead of its effects being just short-lived. The mechanism," he explained, "consists in the ability of caffeine to phosphorylate, and thereby activate, a protein called DARPP-32." Fisone defined the paper's mouth-filling molecule, DARPP-32, by spelling out its descriptive acronym: dopamine and cyclic AMP-regulated phosphoprotein of relative molecular mass 32,000.

"It's very highly expressed in certain cells of the brain," he continued. "A group of structures called the basal ganglia are involved in the control of the body's voluntary movements, among other things. Within the basal ganglia is a population of neurons that express very high levels of DARPP-32. Caffeine is able to act on these cells, and to activate the phosphorylation by modifying DARPP-32. This activation, brought about by caffeine, then in turn amplifies other biochemical reactions, which have an amplification mechanism, we believe, that reinforces and therefore prolongs the effects of caffeine."

New Data Round Out Caffeine's Effects

"Our understanding of caffeine's effects before we wrote this paper," Fisone recalled, "was that the stimulant acts by blocking special receptors. These are usually activated by an endogenous neuromodulator, produced naturally by our brain, called adenosine. By blocking its receptors, caffeine was thought to increase activity by its stimulant effect.

"What we have added today," Fisone continued, "is what happens after caffeine binds to these adenosine receptors, and blocks them. This molecular mechanism accounts for the increase in DARPP-32 phosphorylation. So basically we have found that caffeine, by blocking the adenosine receptors, inhibits an enzyme that normally dephosphorylates DARPP-32, which then in turn acts as an amplifier of the caffeine effect.

"We report a biochemical study to test the mechanism behind this ability of caffeine to stimulate DARPP-32, which was unknown previously. And we have measured the ability of caffeine to work in genetically altered mice, which lack the gene coding for DARPP-32.

"Our initial hypothesis," Fisone recounted, "was that DARPP-32 and its phosphorylation might have been involved in the behavioral, stimulant effects of caffeine. Unfortunately, we couldn't perform these experiments in patients. We had to do them in an animal model. The way in which one can assay the stimulatory effect of caffeine is by measuring how much a mouse moves around in a cage, following the administration by injection of caffeine. So we injected mice systemically with caffeine in a relatively moderate dose. Then we clocked how much these animals moved around in their cage following the caffeine injection.

"After two hours of preliminary exploration, we took them out of their cages for just an instant, injected caffeine, quickly put them back in the same cage. When we injected a mouse with saline physiological solution as a control, took this animal out, injected it and put it back, the animal was a little bit excited the first five minutes. Then its activity returns to very low baseline levels. But when we then injected caffeine, its motor activity increased, and remained so for a few hours after the stimulant dose.

"We then did the same experiment in knockout [KO] mice which cannot produce DARPP," Fisone went on, "then compared their ability to respond to caffeine with the ability of wild-type [WT] normal mice. What we saw was that normal mice had a prolonged response in terms of increase in motor activity, whereas the DARPP-32 knockout mice had a significantly reduced, short-lived response to caffeine. The drug produced the same peak, immediate rapid effect, in both wild-type and knockout animals. But in the KO mice the effect of caffeine declined much more rapidly compared to the WT mice."

Caffeine Findings Abut On Neurodegenerative Ills

"This led us to conclude that the effects of phosphorylation of DARPP-32 on caffeine is not in the very first immediate phase, but is an amplification of a response that takes place when its effects start to decline. At that point, DARPP-32 kicks in with this amplification servomechanism and keeps caffeine acting for a prolonged period of time. This is a positive feedback loop exerted by DARPP-32 on the effects of caffeine.

"DARPP-32 was discovered and studied by Paul Greengard, who is a co-senior author in the article," Fisone recalled. "He's a Nobel Prize winner in physiology or medicine for the year 2000, and discovered DARPP-32 in relation to dopaminergic transmission.

"What we have done now in our ongoing studies," Fisone said, "is to expand his findings that indeed DARPP-32 represents a common denominator for the ability of many different neuroactive substances, not only dopamine, but for instance adenosine and caffeine, and drugs of abuse such as morphine or cannabis. What interests us now is that the basal ganglia are deeply involved in neurodegenerative disorders, in particular those affecting the dopamine system, as in Parkinson's disease. There are followed by a dopamine deficit in the striatal brain regions we have been studying, which," he concluded, "are also affected by caffeine."