By David N. Leff

The most widely used psychoactive drug is not Prozac or Valium, or even cocaine. It's caffeine — as in coffee.

Besides its welcome morning wake-up call, coffee keeps truck drivers and college students alert, with sharpened cognitive capacity. What's more (or less), caffeine, by repeated testing, shows no signs of strong addiction or withdrawal. OD-ing, of course, can cause a racing heart and other nervous symptoms, but these are transient.

In Belgium, there's a breed of mice that stands coffee's familiar stimulatory effects on their heads. Unlike normal mice, which have a healthy sense of curiosity, these rodents stopped exploring their surroundings when fed coffee. All in all, they treated caffeine as a yawn, not a stimulant.

What accounted for this contrarian behavior was the absence of a single gene in their genome, a stretch of DNA that expresses A_2aR, a specific receptor for the nucleoside adenosine. A_2aR is the brain's bull's-eye target for caffeine, which is a central nervous system (CNS) stimulant.

In the brain's striatum, A_2aR is expressed along with receptors for one of the brain's master neurotransmitters, dopamine. It also regulates expression of the precursor gene for enkephalins, which are endorphins thought to mitigate pain perception.

These knockout mice that lack the caffeine receptor are the handiwork of molecular biologist Marc Parmentier and his team at the Free University of Brussels, Institute of Interdisciplinary Research. He is principal author of a paper in today's Nature, dated Aug. 14, 1997, titled: "Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A_2a receptor."

Among other findings, Parmentier told BioWorld Today, this report "demonstrates clearly that the A_2a receptor is mainly involved in caffeine's stimulatory effects. Less expected," he added, "were the behavioral responses in these knockout mice concerning aggressiveness, and to a lesser extent, anxiety. So it suggests that adenosine and this receptor play a role in these behaviors, at least in the mouse, and maybe in humans. That has yet to be confirmed."

By Their Receptors Ye Shall Know Them

After cloning two of adenosine's four receptors in the early 1990s, the Belgium group constructed the A_2aR -minus knockouts by conventional transgenic means, and has studied their behavior patterns over the past 18 months.

Aggressiveness was the salient feature that set these animals apart from their normal littermates. Quantifiable evidence of this antisocial trait was the fact that the knockout animals acquired far more bites than did control animals in the same cage. And male A_2aR -minus mice were much more hostile toward intruders.

How valid is extrapolating this murine experience to the human condition?

"If you can translate that to the human being," Parmentier said, "which is not for sure, it would link aggressiveness to the chronic consumption of coffee. But this has to be tested and demonstrated objectively."

One indirectly objective measure at the molecular level is homology. The murine A_2aR gene, 410 amino acids in length, shares 82.9 percent identity with the human receptor.

Perhaps as a corollary to their aggressiveness, knockout mice appeared to be more anxious than did normal controls.

"Caffeine increases anxiety," the Nature paper noted, "and adenosine-receptor stimulation has been correlated with anxiolytic-like [i.e., soothing] behavior."

To compare the pain responses of their animals, the investigators directed a strong light at the tails of knockout and normal mice. As these appendages heated up, they clocked the time it took a mouse to flick his tail out of pain's way.

Receptor-lacking mice — showing their hypoalgesia (low pain response) — hung in twice as long as wild types before tail-flicking. "The higher nociceptive [pain-perception] threshold," the paper observed, "suggests that the peripheral lack of A_2a receptor predominates over the spinal defect. This," it pointed out, "is in agreement with the observation that acute systemic administration of caffeine results in antinociception."

Cells In Trouble Turn To Adenosine

Adenosine, Parmentier explained, "is essentially a metabolite of ATP [adenosine triphosphate], which is the main energy source of cells. And adenosine synthesis increases when cells are suffering. So it's a way for cells to signal to the surrounding tissue that they are suffering."

He continued: "And adenosine plays a number of roles that protect the cell tissue, for example, in the case of ischemia [oxygen starvation], by promoting vasodilation, by preventing excessive immune response, by averting platelet aggregation.

"All those interventions," he pointed out, "maintain or increase the blood supply, thus reducing the suffering of the cells."

Next, the Belgian team plans to study its knockout animals and their caffeine receptor in the mammalian immune system, as well as adaptation of the CNS to the lack of A2aR. "And," Parmentier said, "we will follow up aggressiveness for sure."

Nor has the caffeine receptor's relevance to industrial drug design escaped their notice.

"The A_2a receptor," they observed in Nature, "is a target for drug design: Antagonists have been considered as treatments for Parkinson's disease, stroke, pain and inflammatory disorders."

"A number of pharmaceutical companies," Parmentier pointed out, "are already developing drugs acting on adenosine receptors. So this is not new. What we consider is that our knockout model is certainly a good tool to study the effect of these drugs, and what can be the expected therapeutic effects in human disease of a drug acting specifically on that receptor."

In an editorial commenting on the Belgian findings, neuroscientist Solomon Snyder, at Johns Hopkins University, in Baltimore, observed: "Pinning down the diverse biological functions of A_2a receptors should provide a route for the pharmaceutical industry to develop related therapeutic agents. One possibility might be new cognitive stimulants."

Snyder concluded: "[Caffeine's] cognition-enhancing actions are well documented. Agents that elicit these beneficial influences . . . may treat cognitive impairment of the elderly." *